Transmembrane serine protease 2 (tmprss2) irna compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., dsRNA agents, targeting the transmembrane serine protein 2 (TMPRSS2) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of a TMPRSS2 gene and to methods of treating or preventing a TMPRSS2-associated disease, e.g., COVID-19, in a subject.

RELATED APPLICATONS

This application is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2021/024076, filed on Mar. 25, 2021, which, in turn, claims the benefit of priority to U.S. Provisional Application No. 63/006,364, filed on Apr. 7, 2020, and U.S. Provisional Application No. 63/050,137, filed on Jul. 10, 2020. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 2, 2022, is named 121301-12603_SL.xml and is 9,189,032 bytes in size.

BACKGROUND OF THE INVENTION

Coronaviruses (CoV) are a large family of viruses that cause diseases in mammals and birds. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases. The name coronavirus is derived from the Latin corona, meaning “crown” or “halo”, which refers to the characteristic appearance reminiscent of a crown or a solar corona around the virions (virus particles) when viewed under two-dimensional transmission electron microscopy, due to the surface covering in club-shaped protein spikes.

The first step of viral infection is viral entry into host cells. The spike (S) protein of coronaviruses facilitates viral entry into target cells. Entry depends on binding of the surface unit, S1, of the S protein to a cellular receptor, which facilitates viral attachment to the surface of target cells. In addition, viral entry into cells requires S protein priming by host cellular proteases, which entails S protein cleavage at the S1/S2 and the S2′ site and allows fusion of viral and cellular membranes, a process driven by the S2 subunit. Previous studies have shown that SARS-CoV-S engages angiotensin-converting enzyme 2 (ACE2) as the entry receptor (Li W, et al., Nature 426, 450-454, 2003) and employs the cellular serine protease TMPRSS2 for S protein priming (Glowacka I., et al., J. Virol. 85, 4122-4134, 2011; Matsuyama S. et al., J. Virol. 84, 12658-12664, 2010; Shulla A., et al., J. Virol. 85, 873-882, 2011). The SARS-CoV-S/ACE2 interface has been elucidated at the atomic level, and the efficiency of ACE2 usage was found to be a key determinant of SARS-CoV transmissibility (Li F, et al., Science 309, 1864-1868, 2005; Li W. et al., EMBO J. 24, 1634-1643, 2005). It has been shown recently that host cell entry of SARS-CoV-2 also depends on the SARS-CoV receptor, ACE2 and that TMPRSS2 is also employed by SARS-CoV-2 for S protein priming (Hoffmann M. at al., Cell 181, 1-10, 2020).

Coronaviruses can cause illness ranging from the common cold to more severe diseases. For example, infections with the human coronavirus strains CoV-229E, CoV-0C43, CoV-NL63 and CoV-HKU1 usually result in mild, self-limiting upper respiratory tract infections, such as a common cold, e.g., runny nose, sneezing, headache, cough, sore throat or fever (Zumla A. et al., Nature Reviews Drug Discovery 15(5): 327-47, 2016; Cheng V. C., et al., Clin. Microbial. Rev. 20: 660-694, 2007; Chan J. F. et al., Clin. Microbial. Rev. 28: 465-522, 2015). Other infections may result in more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV), diseases associated with pneumonia, severe acute respiratory syndrome, kidney failure and death.

MERS-CoV and SARS-CoV have received global attention over the past decades owing to their ability to cause community and health-care-associated outbreaks of severe infections in human populations. MERS-CoV is a viral respiratory disease that was first reported in Saudi Arabia in 2012 and has since spread to more than 27 other countries, according to the World Health Organization (de Groot, R. J. et al., J. Virol. 87: 7790-7792, 2013). SARS was first reported in Asia in 2003, and quickly spread to about two dozen countries before being contained after about four months (Lee N. et al., N. Engl. J. Med. 348: 1986-1994, 2003; Peiris J. S. et al., Lancet 36: 1319-1325, 2003). Detailed investigations found that SARS-CoV was transmitted from civet cats to humans and MERS-CoV from dromedary camels to humans (Cheng V. C., et al., Clin. Microbial. Rev. 20: 660-694, 2007; Chan J. F. et al., Clin. Microbial. Rev. 28: 465-522, 2015).

A recent outbreak of respiratory disease caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan City, China. This disease, named by the World Health Organization as coronavirus disease 2019 (“COVID-19”), presents a major threat to public health worldwide. As of Mar. 26, 2020, there were more than 529,000 confirmed cases and 23,000 deaths across the world.

Coronaviruses pose major challenges to clinical management because many questions regarding transmission and control remain unanswered. Moreover, there is currently no vaccine to prevent infections by coronavirus, and there are no specific antiviral treatments available or proven to be effective to treat or prevent coronavirus infection in subjects. Given the critical role that TMPRSS2 plays in the first step of viral infection, TMPRSS2 constitutes a target for antiviral treatment.

Accordingly, there exists an immediate need for an agent that can selectively and efficiently silence the TMPRSS2 gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target TMPSS2 gene.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides RNAi agent compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcipts of a gene encoding Transmembrane Serine Protease 2 (TMPRSS2). The TMPRSS2 gene may be within a cell, e.g., a cell within a subject, such as a human The present disclosure also provides methods of using the RNAi agent compositions of the disclosure for inhibiting the expression of a TMPRSS2 gene or for treating a subject who would benefit from inhibiting or reducing the expression of a TMPRSS2 gene, e.g., a subject having a TMPRSS2-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV), or a subject at risk of developing a coronavirus infection, e.g., during an epidemic or pandemic.

Accordingly, in one aspect, the instant disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a Transmembrane Serine Protease 2 (TMPRSS2) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:6; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a TMPRSS2 gene in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region complementary to part of an mRNA encoding a TMPRSS2 gene (SEQ ID NO:1), wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In yet another aspect, the present invention provides a double stranded RNAi agent for inhibiting expression of a a TMPRSS2 gene in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-3, wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-3.

In one embodiment, both the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent.

In one embodiment, the lipophilic moiety is conjugated via a linker or a carrier.

In one embodiment, lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.

In one embodiment, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.

In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In one embodiment, no more than five of the sense strand nucleotides and no more than five of the nucleotides of the antisense strand are unmodified nucleotides

In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selected from the group a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, a 2′-O hexadecyl nucleotide, a nucleotide comprising a 2′-phosphate, a cytidine-2′-phosphate nucleotide, a guanosine-2′-phosphate nucleotide, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide, a a 5′-vinyl phosphonate (VP), a 2′-deoxyadenosine-3′-phosphate nucleotide, a 2′-deoxycytidine-3′-phosphate nucleotide, a 2′-deoxyguanosine-3′-phosphate nucleotide, a 2′-deoxythymidine-3′-phosphate nucleotide, a 2′-deoxyuridine nucleotide, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

In another embodiment, modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In another embodiment, the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In yet another embodiment, the modifications on the nucleotides are 2′-O-methyl modifications, 2′-deoxy-modifications, 2′fluoro modifications, 5′-vinyl phosphonate (VP) modification, and 2′-O hexadecyl nucleotide modifications.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

In one embodiment, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages.

In one embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide.

In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

The double stranded region may be 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length;19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

Each strand of the dsRNA agent may be has 19-30 nucleotides in length; 19-23 nucleotides in length; or 21-23 nucleotides in length.

In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

In one embodiment, the internal positions include all positions except the terminal two positions from each end of the at least one strand.

In another embodiment, the internal positions include all positions except the terminal three positions from each end of the at least one strand.

In another embodiment, the internal positions exclude a cleavage site region of the sense strand.

In yet another embodiment, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand.

In one embodiment, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand.

In one embodiment, the internal positions exclude a cleavage site region of the antisense strand.

In one embodiment, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand.

In one embodiment, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′ end of each strand.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

In one embodiment, the positions in the double stranded region exclude a cleavage site region of the sense strand.

In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

In one embodiment, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5′-end of the strand.

In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.

In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

In one embodiment, the lipophilic moiety or a targeting ligand is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In one embodiment, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

In one embodiment, the dsRNA agent further comprises a targeting ligand that targets a liver tissue.

In one embodiment, the targeting ligand is a GalNAc conjugate.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand.

In one embodiment, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

The present invention further provides cells, pharmaceutical compositions for inhibiting expression of a TMPRSS2 gene, and pharmaceutical composition comprising a lipid formulation comprising the dsRNA agent of the invention.

In one aspect, the present invention provides a method of inhibiting expression of a TMPRSS2 gene in a cell. The method includes contacting the cell with the dsRNA agent of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TMPRSS2 gene, thereby inhibiting expression of the TMPRSS2 gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human

In one embodiment, the expression of the TMPRSS2 gene is inhibited by at least 50%.

In one aspect, the present invention provides a method of inhibiting entry of a coronavirus into a cell. The method includes contacting the cell with the dsRNA agent of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the TMPRSS2 gene, thereby inhibiting entry of the coronavirus into the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human

In one embodiment, the expression of the TMPRSS2 gene is inhibited by at least 50%.

In one aspect, the present invention provides a method of inhibiting replication of a coronavirus in a cell. The method includes contacting the cell with the dsRNA agent of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the TMPRSS2 gene, thereby inhibiting replication of the coronavirus in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human

In one embodiment, the expression of the TMPRSS2 gene is inhibited by at least 50%.

In another aspect, the present invention provides a method of inhibiting priming of a coronavirus S protein in a cell. The method includes contacting the cell with the dsRNA agent of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of an mRNA transcript of a TMPRSS2 gene, thereby inhibiting priming of a coronavirus S protein in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human

In one embodiment, the expression of the TMPRSS2 gene is inhibited by at least 50%.

In one aspect, the present invention provides a method of treating a TNPRSS2-associate disorder, e.g., a subject having a coronavirus infection or at risk of developing or at risk of having a coronavirus infection. The method includes administering to the subject a therapeutically effective amount of the dsRNA agent of the invention, or the pharmaceutical composition of the invention, thereby treating the subject.

In one embodiment, the subject is a human

In one embodiment, the subject having the coronavirus infection is infected with a severe acute respiratory syndrome (SARS) virus, a Middle East respiratory syndrome (MERS) virus, or a severe acute respiratory syndrome 2 (SARS-2) virus.

In one embodiment, the subject at risk of developing a coronavirus infection, e.g., an infection caused by severe acute respiratory syndrome (SARS) virus, a Middle East respiratory syndrome (MERS) virus, or a severe acute respiratory syndrome 2 (SARS-2) virus, is a subject in an epidemic or pandemic.

In one embodiment, treating comprises amelioration of at least on sign or symptom of the disease.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, the administration of the dsRNA is pulmonary system administration.

In one embodiment, the pulmonary system administration is oral inhalation or intranasally.

In one embodiment, the method reduces the expression of an TMPRSS2 gene in a pulmonary system tissue, e.g., a nasopharynx tissue, an oropharynx tissue, a laryngopharynx tissue, a larynx tissue, a trachea tissue, a carina tissue, a bronchi tissue, a bronchiole tissue, or an alveoli tissue.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the method further comprises administering to the subject an additional agent or a therapy suitable for treatment or prevention of a coronavirus-associated disorder.

In one embodiment, the additional therapeutic agent is selected from the group consisting of an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.

The present invention is further illustrated by the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a TMPRSS2 gene. The TMPRSS2 gene may be within a cell, e.g., a cell within a subject, such as a human The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (a TMPRSS2 gene) in mammals. The present disclosure also provides methods of using the RNAi compositions of the disclosure for inhibiting the expression of a TMPRSS2 gene for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a TMPRSS2 gene, e.g., a TMPRSS2-associated disorder, e.g., a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV), or a subject at risk of a coronavirus infection, e.g., infection by Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV), e.g., during an epidemic or pandemic

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a TMPRSS2 gene. In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 21-23 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a TMPRSS2 gene.

In certain embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA of a TMPRSS2 gene. In some embodiments, such iRNA agents having longer length antisense strands preferably may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation of the TMPRSS2 mRNAs in mammals Thus, methods and compositions including these iRNAs are useful for treating a subject having a TMPRSS2-associated disorder, e.g., a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV) or treating a subject at risk of a TMPRSS2-associate disorder, e.g., a subject at risk of a coronavirus infection, e.g., infections resulting in Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV), e.g., during an epidemic or pandemic.

In certain embodiments, the administration of the dsRNA to a subject results in an improvement in viral load, of lung function, or a stoppage or reduction of the rate of loss of lung function, reduction of fever, reduction of cough.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a TMPRSS2 gene s as well as compositions, uses, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of a TMPRSS2 gene, e.g., subjects susceptible to or diagnosed with a TMPRSS2-associated disorder.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

As used herein, the term “coronavirus,”(“CoV”; subfamily Coronaviridae, family Coronaviridae, order Nidovirales), refers to a group of highly diverse, enveloped, positive-sense, single-stranded RNA viruses that cause respiratory, enteric, hepatic and neurological diseases of varying severity in a broad range of animal species, including humans. Coronaviruses are subdivided into four genera: Alphacoronavirus, Betacoronavirus (13CoV), Gammacoronavirus and Deltacoronavirus.

Any coronavirus that infects humans and animals is encompassed by the term “coronavirus” as used herein. Exemplary coronaviruses encompassed by the term include the coronaviruses that cause a common cold-like respiratory illness, e.g., human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), and human coronavirus HKU1 (HCoV-HKU1); the coronavirus that causes avian infectious bronchitis virus (IBV); the coronavirus that causes murine hepatitis virus (MHV); the coronavirus that causes porcine transmissible gastroenteritis virus PRCoV; the coronavirus that causes porcine respiratory coronavirus and bovine coronavirus; the coronavirus that causes Severe Acute Respiratory Syndrome (SARS), the coronavirus that causes the Middle East respiratory syndrome (MERS), and the coronavirus that causes Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19).

The coronavirus (CoV) genome is a single-stranded, non-segmented RNA genome, which is approximately 26-32 kb. It contains 5′-methylated caps and 3′-polyadenylated tails and is arranged in the order of 5′, replicase genes, genes encoding structural proteins (spike glycoprotein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)), polyadenylated tail and then the 3′ end. The partially overlapping 5′-terminal open reading frame la/b (ORF1a/b) is within the 5′ two-thirds of the CoV genome and encodes the large replicase polyprotein 1a (pp1a) and pplab. These polyproteins are cleaved by papain-like cysteine protease (PLpro) and 3C-like serine protease (3CLpro) to produce non-structural proteins, including RNA-dependent RNA polymerase (RdRp) and helicase (Hel), which are important enzymes involved in the transcription and replication of CoVs. The 3′ one-third of the CoV genome encodes the structural proteins (S, E, M and N), which are essential for virus—cell-receptor binding and virion assembly, and other non-structural proteins and accessory proteins that may have immunomodulatory effects. (Peiris J S., et al.,2003, Nat. Med. 10 (Suppl. 12): 88-97).

As a coronavirus is a positive-sense, single-stranded RNA virus having a 5′ methylated cap and a 3′ polyadenylated tail, once the virus enters the cell and is uncoated, the viral RNA genome attaches to the host cell's ribosome for direct translation. The host ribosome translates the initial overlapping open reading frame of the virus genome and forms a long polyprotein. The polyprotein has its own proteases which cleave the polyprotein into multiple nonstructural proteins.

A number of the nonstructural proteins coalesce to form a multi-protein replicase-transcriptase complex (RTC). The main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). It is directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease non-structural protein for instance provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks.

One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA. The other important function of the complex is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. This is followed by the transcription of these negative-sense subgenomic RNA molecules to their corresponding positive-sense mRNAs

The replicated positive-sense genomic RNA becomes the genome of the progeny viruses.

As use herein, the term “severe acute respiratory syndrome coronavirus” or “SARS-CoV”, refers to a coronavirus that was first discovered in 2003, which causes severe acute respiratory syndrome (SARS). SARS-CoV represents the prototype of a new lineage of coronaviruses capable of causing outbreaks of clinically significant and frequently fatal human disease. The complete genome of SARS-CoV has been identified, as well as common variants thereof. The genome of SARS-CoV is a 29,727-nucleotide polyadenylated RNA, has 11 open reading frames, and 41% of the residues are G or C. The genomic organization is typical of coronaviruses, with the characteristic gene order (5′-replicase (rep), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′ and short untranslated regions at both termini The SARS-CoV rep gene, which comprises about two-thirds of the genome, is predicted to encode two polyproteins that undergo co-translational proteolytic processing. There are four open reading frames (ORFs) downstream of rep that are predicted to encode the structural proteins, S, E, M and N. The hemagglutinin-esterase gene, which is present between ORFlb and S in group 2 and some group 3 coronaviruses was not found.

The amino acid and complete coding sequences of the SARS-CoV genomes are known may be found in for example, GenBank Accession Nos. AY502923.1; AP006559.1; AP006558.1; AY313906.1; AY345986.1; AY502931.1; AY282752.2; AY559097.1; AY559081.1; DQ182595.1; AY291451.1; AY568539.1; AY613947.1; and AY390556.1, the entire contents of each of which are incorporated herein by reference.

The term “SARS-CoV,” as used herein, also refers to naturally occurring RNA sequence variations of the SARS-CoV genome.

As use herein, the term “the Middle East respiratory syndrome coronavirus” or “MERS-CoV”, refers to a coronavirus that causes the Middle East respiratory syndrome (MERS), which was first identified in 2012. MERS-CoV is closely related to severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). Clinically similar to SARS, MERS-CoV infection leads to severe respiratory illness with renal failure.

The amino acid and complete coding sequences of the MERS-CoV genomes are known and may be found in for example, GenBank Accession Nos. MK462243.1; MK462244.1; MK462245.1; MK462246.1; MK462247.1; MK462248.1; MK462249.1; MK462250.1; MK462251.1; MK462252.1; MK462253.1; MK462254.1; MK462255.1; MK462256.1; MK483839.1; and MH822886.1, the entire contents of each of which are incorporated herein by reference.

The term “MERS-CoV,” as used herein, also refers to naturally occurring RNA sequence variations of the MERS-CoV genome.

As use herein, the terms “severe acute respiratory syndrome coronavirus 2,” “SARS-CoV-2,” “2019-nCoV,” refer to the novel coronavirus that caused a pneumonia outbreak first reported in Wuhan, China in December 2019 (“COVID-19”). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that SARS-CoV-2 was most closely related (89.1% nucleotide similarity similarity) to SARS-CoV.

The amino acid and complete coding sequences of the SARS-CoV-2 genomes are known and may be found in for example, the GISAID EpiCoVTM Database (db.cngb.org/gisaid/), including Accession nos. EPI_ISL_402119; EPI_ISL_402120; EPI_ISL_402121; EPI_ISL_402123; EPI_ISL_402124; EPI_ISL_402125; EPI_ISL_402127; EPI_ISL_402128; EPI_ISL_402129; EPI_ISL_402130; EPI_ISL_402132; EPI_ISL_403928; EPI_ISL_403929; EPI_ISL_403930; EPI_ISL_403931; EPI_ISL_403932; EPI_ISL_403933; EPI_ISL_403934; EPI_ISL_403935; EPI_ISL_403936; EPI_ISL_403937; EPI_ISL_403962; EPI_ISL_404228; EPI_ISL_404253; and EPI_ISL_404895, the entire contents of wach of which are incorporated herein by reference.

The term “SARS-CoV-2,” as used herein, also refers to naturally occurring RNA sequence variations of the SARS-CoV-2 genome.

Additional examples of coronavirus genomes and mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, the term “Transmembrane Serine Protease 2,” used interchangeably with the term “TMPRSS2,” refers to the well-known gene and polypeptide, also known in the art as Serine Protease 10, and SS10. The term “TMPRSS2” includes human TMPRSS2, the amino acid and nucleotide sequences of which may be found in, for example, GenBank Accession No. NM_005656.4 (GI:1581462622; SEQ ID NO:1) and GenBank Accession No. NM_001135099.1 (GI: 227499989; SEQ ID NO: 2); mouse TMPRSS2, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. NM_015775.2 (GI: 34328225, SEQ ID NO: 3); and rat TMPRSS2, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession: NM_130424.3 (GI: 829708433; SEQ ID NO: 4).

The term “TMPRSS2” also includes Macaca fascicularis TMPRSS2, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. XM_005548643.2 (GI: 982237460; SEQ ID NO: 5)

Additional examples of TMPRSS2 mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary TMPRSS2 nucleotide sequences may also be found in SEQ ID NOs:1-10. SEQ ID NOs:6-10 are the reverse complement sequences of SEQ ID NOs:1-5, respectively.

Further information on TMPRSS2 is provided, for example in the NCBI Gene database at www.ncbi.nlm.nih.gove/gene/7113.

The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

The terms “transmembrane serine protease 2” and “TMPRSS2,” as used herein, also refers to naturally occurring DNA sequence variations of the TMPRSS2 gene. Numerous sequence variations within the TMPRSS2 gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov/snp?LinkName=gene_snp&from_uid=1773), the entire contents of which is incorporated herein by reference as of the date of filing this application.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TMPRSS2 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TMPRSS2 gene. In one embodiment, the target sequence is within the protein coding region of the TMPRSS2 gene. In another embodiment, the target sequence is within the 3′ UTR of the TMPRSS2 gene.

The target sequence may be from about 19-36 nucleotides in length, e.g., preferably about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulates, e.g., inhibits, the expression of a TMPRSS2 gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a TMPRSS2 mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, a “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a TMPRSS2 mRNA sequence. In some embodiments of the disclosure, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide—which is acknowledged as a naturally occurring form of nucleotide—if present within a RNAi agent can be considered to constitute a modified nucleotide.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5′-end of first strand is linked to the 3′-end of the second strand or 3′-end of first strand is linked to 5′-end of the second strand. When the two strands are linked to each other at both ends, 5′-end of first strand is linked to 3′-end of second strand and 3′-end of first strand is linked to 5′-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiemtns, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleosidic linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.

Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3′, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as “shRNA” herein.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a TMPRSS2 mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In one embodiment, an RNAi agent of the invention is a dsRNA agent, each strand of which comprises 19-23 nucleotides that interacts with a TMPRSS2 mRNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a TMPRSS2 mRNA sequence to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a TMPRSS2 mRNA sequence.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a TMPRSS2 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the RNAi agent.

In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand double stranded RNA agent of the invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region.

Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a TMPRSS2 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a TMPRSS2 gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a TMPRSS2 gene is important, especially if the particular region of complementarity in a TMPRSS2 gene is known to vary.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within a RNAi agent, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest or target sequence (e.g., an mRNA encoding TMPRSS2). For example, a polynucleotide is complementary to at least a part of a TMPRSS2 RNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding TMPRSS2.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target TMPRSS2 sequence.

In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target TMPRSS2 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1-5 for TMPRSS2, or a fragment of SEQ ID Nos: 1-5, such as about 85%, about 90%, or about 95% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target TMPRSS2 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2 and 3, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2 and 3, such as about 85%, about 90%, or about 95% complementary.

In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target TMPRSS2 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 6-10, or a fragment of any one of SEQ ID NOs: 6-10, such as about 85%, about 90%, or about 95% complementary.

In some embodiments, an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target TMPRSS2 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of any one of Tables 2 and 3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2 and 3, such as about 85%, about 90%, or about 95% complementary.

In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21-nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3′-end.

In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

In one embodiment, at least partial suppression of the expression of a TMPRSS2 gene, is assessed by a reduction of the amount of TMPRSS2 mRNA which can be isolated from or detected in a first cell or group of cells in which a TMPRSS2 gene is transcribed and which has or have been treated such that the expression of a TMPRSS2 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In one embodiment, inhibition of expression is determined by the dual luciferase method in Example 1 wherein the RNAi agent is present at 10 nM.

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, via inhalation, intranasal administration, or intratracheal administration, by injecting the RNAi agent into or near the tissue where the cell is located, e.g., the pulmonary system, or by injecting the RNAi agent into another area, or to the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT Publication No. WO 2019/217459, the entire contents of which is incorporated herein by reference, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the pulmonary system. In some embodiments, the RNAi agent may contain or be coupled to a ligand, e.g., one or more GalNAc derivatives as described below, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the liver. In other embodiments, the RNAi agent may contain or be coupled to a lipophilic moiety or moieties and one or more GalNAc derivatives. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, logK_(ow), where K_(ow) is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its logK_(ow) exceeds 0. Typically, the lipophilic moiety possesses a logK_(ow) exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logK_(ow) of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logK_(ow) of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK_(ow)) value of the lipophilic moiety.

Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.

In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT Publication No. WO 2019/217459. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a rNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In a preferred embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit from reduction in TMPRSS2 expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in TMPRSS2 expression; a human having a disease, disorder, or condition that would benefit from reduction in TMPRSS2 expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in TMPRSS2 expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with TMPRSS2 expression or TMPRSS2 protein production, e.g., a TMPRSS2-associated disease, e.g., a coronavirus-associated disease, e.g., COVID-19. Treatment also includes a reduction of one or more sign or symptoms associated with unwanted TMPRSS2 expression; diminishing the extent of unwanted TMPRSS2 activation or stabilization; amelioration or palliation of unwanted TMPRSS2 activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of TMPRSS2 in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of TMPRSS2 in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., viral load, blood oxygen level, white blood cell count, kidney function, liver function . As used here, “lower” in a subject can refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production or viral replication in a subject.

The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a TMPRSS2-associated disease towards or to a level in a normal subject not suffering from a TMPRSS2-associated disease. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder, or condition thereof, that would benefit from a reduction in expression of a TMPRSS2 gene or production of a TMPRSS2 protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a TMPRSS2-associated disease, e.g., COVID-19. The failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition, e.g., pneumonia (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “TMPRSS2-associated disease,” is a disease or disorder that would benefit from reduction in the expression or activity of TMPRSS2. Such TMPRSS2-associated diseases include a coronavirus-associated disease.

The term “coronavirus-associated disease,” is a disease or disorder that is caused by, or associated with a coronavirus infection, coronavirus genome expression or coronavirus protein production. The term “coronavirus-associated disease” includes a disease, disorder or condition that would benefit from a decrease in coronavirus S protein priming, viral genome expression, cellular (viral) entry, viral replication, or viral protein activity.

Non-limiting examples of coronavirus-associated diseases include, for example, disease or disorders caused by infection with human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS), the Middle East respiratory syndrome coronavirus (MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19). The symptoms for a coronavirus-associated disease depend on the type of coronavirus and how serious the infection is. Patients with a mild to moderate upper-respiratory infection may develop symptoms such as runny nose, sneezing, headache, cough, sore throat, fever, or short of breath. In more severe cases, coronavirus infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death. Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a coronavirus-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having a TMPRSS2-associated disorder, e.g., a coronavirus-associated disorder, such as COVID-19, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, bronchial fluids, sputum, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from a nasal swab. In certain embodiments, samples may be derived from a throat swab. In certain embodiments, samples may be derived from the lung, or certain types of cells in the lung. In some embodiments, the samples may be derived from the bronchioles. In some embodiments, the samples may be derived from the bronchus. In some embodiments, the samples may be derived from the alveoli. In other embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) derived from the subject. In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma or serum derived therefrom. In further embodiments, a “sample derived from a subject” refers to pulmonary tissue (or subcomponents thereof) derived from the subject.

II. RNAi Agents of the Disclosure

Described herein are RNAi agents which inhibit the expression of a TMPRSS2 gene. In one embodiment, the RNAi agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a TMPRSS2 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human, e.g., a subject having a TMPRSS2-associated disorder, e.g., a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV). The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of a target RNA, e.g., an mRNA formed in the expression of a TMPRSS2 gene. The region of complementarity is about 15-30 nucleotides or less in length. Upon contact with a cell expressing the TMPRSS2 gene, the RNAi agent inhibits the expression of the TMPRSS2 gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In preferred embodiments, inhibition of expression is by at least 50% as assayed by the Dual-Glo lucifierase assay in Example 1 where the siRNA is at a 10 nM concentration.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. For example, the target sequence can be derived from the sequence of an mRNA formed during the expression of a TMPRSS2 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

In some embodiments, the dsRNA is 15 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, a RNAi agent useful to target TMPRSS2 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments, longer, extended overhangs are possible.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

An siRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

An siRNA can be made by separately synthesizing a single stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, after which the component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given siRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the siRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to produce a discrete siRNA species. The complementary of the species to a TMPRSS2 gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

In one embodiment, RNA generated is carefully purified to remove endsiRNA is cleaved in vitro into siRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g., a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9 and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsiRNA cleavage generally produces a plurality of siRNA species, each being a particular 21 to 23 nucleotide fragment of a source dsiRNA molecule. For example, siRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsiRNA molecule may be present.

Regardless of the method of synthesis, the siRNA preparation can be prepared in a solution (e.g., an aqueous or organic solution) that is appropriate for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process.

In one aspect, a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for TMPRSS2 may be selected from the group of sequences provided in any one of Tables 2 and 3, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences of any one of Tables 2 and 3. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a TMPRSS2 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2 and 3, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2 and 3 for TMPRSS2.

In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences provided herein are described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth in any one of Tables 2 and 3 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. One or more lipophilic ligands or one or more GalNAc ligands can be included in any of the positions of the RNAi agents provided in the instant application.

The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a TMPRSS2 gene by not more than 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence using the in vitro assay with Cos7 and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure.

In addition, the RNAs described herein identify a site(s) in a TMPRSS2 transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features RNAi agents that target within this site(s). As used herein, a RNAi agent is said to target within a particular site of an RNA transcript if the RNAi agent promotes cleavage of the transcript anywhere within that particular site. Such a RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a TMPRSS2 gene.

An RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a TMPRSS2 gene generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a TMPRSS2 gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a TMPRSS2 gene is important, especially if the particular region of complementarity in a TMPRSS2 gene is known to mutate.

III. Modified RNAi Agents of the Disclosure

In one embodiment, the RNA of the RNAi agent of the disclosure e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In preferred embodiments, the RNA of an RNAi agent of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of an RNAi agent of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of an RNAi agent of the disclosure are modified. RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides.

The nucleic acids featured in the disclosure can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified RNAi agent will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, e.g., sodium salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂-[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(·n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a RNAi agent, or a group for improving the pharmacodynamic properties of a RNAi agent, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′ CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of a RNAi agent, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. RNAi agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An RNAi agent of the disclosure can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

An RNAi agent of the disclosure can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

An RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative US Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

An RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the —C3′ and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO 2011/005861.

Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified RNAi agents Comprising Motifs of the Disclosure

In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic ligand, e.g., a C16 ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.

Accordingly, the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a targetgenome or gene (i.e., a TMPRSS2 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15-30 nucleotides in length. For example, each strand may be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In preferred embodiments, the duplex region is 19-21 nucleotide pairs in length.

In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In preferred embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-O-methyl, thymidine (T), and any combinations thereof.

For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand. When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16ligand).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complemenatary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^(st) nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^(st) paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adajacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two, or three nucleotides in the duplex region.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

5′ n_(p)-N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q) 3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_(a) or Nb comprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11,12 or 11, 12, 13) of — the sense strand, the count starting from the ^(1st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′ n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′  (Ib);

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q) 3′  (Ic); or

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′   (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′   (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′ n_(q′)-N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′ 3′  (II)

wherein:

k and l are each independently 0 or 1

p′ and q′ are each independently 0-6;

-   each N_(a)′ independently represents an oligonucleotide sequence     comprising 0-25 modified nucleotides, each sequence comprising at     least two differently modified nucleotides; -   each N_(b)′ independently represents an oligonucleotide sequence     comprising 0-10 modified nucleotides; -   each n_(p)′ and n_(q)′ independently represent an overhang     nucleotide; -   wherein N_(b)′ and Y′ do not have the same modification; and -   X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of     three identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ or N_(b)′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14 ; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′ n_(q′)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n_(p′) 3′  (IIb);

5′ n_(q′)-N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n_(p′) 3′  (IIc); or

5′ n_(q′)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p′) 3′   (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b 'is) 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′ n_(p′)-N_(a′)—Y′Y′Y′—N_(a′)-n_(q′) 3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′ n_(p)-N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q) 3′

antisense: 3′ n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′ 5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each Nb and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

5′ n_(p)-N_(a)—Y Y Y—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)′n_(q)′ 5′  (IIIa)

5′ n_(p)-N_(a)—Y Y Y—Z Z Z—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)′n_(q)′ 5′  (IIIb)

5′ n_(p)-N_(a)—X X X—N_(b)—Y Y Y—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′  (IIIc)

5′ n_(p)-N_(a)—X X X—N_(b)—Y Y Y—N_(b)—Z Z Z—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)—Z′Z′Z′—N_(a)-n_(q)′ 5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (Mb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′ independently comprises modifications of alternating pattern.

In one embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications , n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications , n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, optionally attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications , n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the entire contents of each of which are hereby incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:

A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:

E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

Exemplified abasic modifications include, but are not limited to the following:

Wherein R═H, Me, Et or OMe; R′═H, Me, Et or OMe; R″═H, Me, Et or OMe

wherein B is a modified or unmodified nucleobase.

Exemplified sugar modifications include, but are not limited to the following:

wherein B is a modified or unmodified nucleobase.

In some embodiments the thermally destabilizing modification of the duplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent or at least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′, or O4′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W—C H-bonding to complementary base on the target mRNA, such as:

More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.

The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more a-nucleotide complementary to the base on the target mRNA, such as:

wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂ or O-alkyl.

Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:

The alkyl for the R group can be a C₁-C₆alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.

In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.

In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2′-fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one 2′-fluoro nucleotide adjacent to the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four 2′-fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5′-end of the antisense strand. Preferably, the 2 nt overhang is at the 3′-end of the antisense.

In some embodiments, the dsRNA molecule of the disclosure comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1), positions 1 to 23 of said sense strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complimentary to positions 14-17 of the 5′-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12-30 nucleotide pairs in length.

In some embodiments, the dsRNA molecule of the disclosure comprises a sense and antisense strands, wherein said dsRNA molecule comprises a sense strand having a length which is at least 25 and at most 29 nucleotides and an antisense strand having a length which is at most 30 nucleotides with the sense strand comprises a modified nucleotide that is susceptible to enzymatic degradation at position 11 from the 5′end, wherein the 3′ end of said sense strand and the 5′ end of said antisense strand form a blunt end and said antisense strand is 1-4 nucleotides longer at its 3′ end than the sense strand, wherein the duplex region which is at least 25 nucleotides in length, and said antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of said antisense strand length to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said antisense strand, thereby reducing expression of the target gene in the mammal, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. e.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′ aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region.

For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.

In some embodiments, 5′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 4′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4′-O-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4′-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

In another embodiment, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2 and 3. These agents may further comprise a ligand, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both of one of more lipophilic moieties and one or more GalNAc derivatives.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides alkyl & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.

In certain embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an a-helical agent and can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:11). An RFGF analogue (e g , amino acid sequence AALLPVLLAAP (SEQ ID NO:12)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:13)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:14)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and tri-saccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate comprises a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments the GalNAc conjugate is conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:

In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands, even if such GalNAc ligands are currently projected to be of limited value for the preferred pulmonary system delivery route(s) of the instant disclosure.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 5′ end of the sense strand of a dsRNA agent, or the 5′ end of one or both sense strands of a dual targeting RNAi agent as described herein. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In certain embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(P)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-S—P(O)(Rk)-S—, —O—P(S)(Rk)-S. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid cleavable linking groups

In certain embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Cleavable Linking Groups

In certain embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleavable Linking Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andRa is H or amino acid side chain.Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XLIX):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. Patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928;5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNA agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of an RNAi Agent of the Disclosure

The delivery of a RNAi agent of the disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a TMPRSS2-associated disorder, e.g., a coronavirus-associated disorder, e.g., a subject having or at risk of developing of at risk of having a coronavirus infection, e.g., a subject having or at risk of developing or at risk of having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV)), can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an RNAi agent of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an RNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a RNAi agent of the disclosure (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. The non-specific effects of an RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies have shown successful knockdown of gene products when an RNAi agent is administered locally. For example, pulmonary delivery, e.g., inhalation, of a dsRNA, e.g., SOD1, has been shown to effectively knockdown gene and protein expression in lung tissue and that there is excellent uptake of the dsRNA by the bronchioles and alveoli of the lung. Intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M I et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were also both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et a.l (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya,Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such seed region destabilization). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a RNAi agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making and administering cationic-RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Certain aspects of the instant disclosure relate to a method of reducing the expression of a TMPRSS2 gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In one embodiment, the cell is a hepatic cell, optionally a hepatocyte. In one embodiment, the cell is an extrahepatic cell, optionally a pulmonary cell.

Another aspect of the disclosure relates to a method of reducing the expression and/or activity of a TMPRSS2 gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.

Another aspect of the disclosure relates to a method of treating a subject having a TMPRSS2-associated disorder orat risk of having or at risk of developing a TMPRss2-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject. In some embodiments, the TMPRSS2-associated disorder comprises a coronavirus-associated disorder. Non-limiting examples of coronavirus-associated diseases include, for example, Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV).

In one embodiment, the double-stranded RNAi agent is administered subcutaneously.

In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration, or oral inhalative administration.

In one embodiment, the double-stranded RNAi agent is administered intranasally.

By pulmonary system administration, e.g., intranasal administration or oral inhalative administration, of the double-stranded RNAi agent, the method can reduce the expression of an TMPRSS2 target gene in a pulmonary system tissue, e.g., a nasopharynx tissue, an oropharynx tissue, a laryngopharynx tissue, a larynx tissue, a trachea tissue, a carina tissue, a bronchi tissue, a bronchiole tissue, or an alveoli tissue.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. A composition that includes a RNAi agent can be delivered to a subject by a variety of routes. Exemplary routes include pulmonary system, intravenous, intraventricular, topical, rectal, anal, vaginal, nasal, and ocular.

The RNAi agents of the disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of RNAi agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the RNAi agent in powder or aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA.

Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.

In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

Pulmonary System Administration

In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration. The pulmonary system includes the upper pulmonary system and the lower pulmonary system. The upper pulmonary system includes the nose and the pharynx. The pharynx includes the nasopharynx, oropharynx, and laryngopharynx. The lower pulmonary system includes the larynx, trachea, carina, bronchi, bronchioles, and alveoli.

Pulmonary system dministration may be intranasal administration or oral inhalative administration. Such administration permits both systemic and local delivery of the double stranded RNAi agents of the invention.

Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization. Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays.

Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system. Suitable dosage forms for oral inhalative administration include powders and solutions. Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, N.Y.) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet nebulizers are driven by compressed air. Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode. The holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross-section size on the side from where the droplets emerge. Depending on the therapeutic application, the hole sizes and number of holes can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs. The amount of RNAi agent for pulmonary system administration may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, preferably 50 μg to 1500 μg, more preferably 100 μg to 1000 μg.

Vector Encoded RNAi Agents of the Disclosure

RNAi agents targeting the TMPRSS2 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and U.S. 6,054,299). Expression is preferablysustained (months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of a RNAi agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a RNAi agent as described herein. Delivery of RNAi agent expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of a RNAi agent will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells. Other aspects to consider for vectors and constructs are known in the art.

VI. Pharmaceutical Compositions of the Invention

The present disclosure also includes pharmaceutical compositions and formulations which include the RNAi agents of the disclosure. In one embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent are useful for treating a subject who would benefit from inhibiting or reducing the expression of a TMPRSS2 gene, e.g., a subject having a TMPRSS2 —associated disorder, e.g, a coronavirus-associated disorder, e.g., a subject having or at risk of having or at risk of developing a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV). Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for direct delivery into the pulmonary system by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal delivery. Another example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery.

In some embodiments, the pharmaceutical compositions of the invention are pyrogen free or non-pyrogenic.

The pharmaceutical compositions of the disclosure may be administered in dosages sufficient to inhibit expression of a TMPRSS2 gene. In general, a suitable dose of an RNAi agent of the disclosure will be a flat dose in the range of about 0.001 to about 200.0 mgabout once per month to about once per year, typically about once per quarter (i.e., about once every three months) to about once per year, generally a flat dose in the range of about 1 to 50 mg about once per month to about once per year, typically about once per quarter to about once per year.

After an initial treatment regimen (e.g., loading dose), the treatments can be administered on a less frequent basis.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.

Advances in mouse genetics have generated a number of mouse models for the study of various TMPRSS2-associated diseases that would benefit from reduction in the expression of TMPRSS2. Such models can be used for in vivo testing of RNAi agents, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, the mouse models described elsewhere herein.

The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary system administration by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The RNAi agents can be delivered in a manner to target a particular tissue, such as the liver, the lung (e.g., bronchioles, alveoli, or bronchus of the lung), or both the liver and lung.

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the RNAi agents featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). RNAi agents featured in the disclosure can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi agents can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. RNAi Agent Formulations Comprising Membranous Molecular Assemblies

A RNAi agent for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the RNAi agent composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the RNAi agent are delivered into the cell where the RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the RNAi agent to particular cell types.

A liposome containing an RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid or phosphatidylcholine or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S. T. P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85,:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No.4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

Liposomes that include RNAi agents can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present disclosure are described in PCT publication No. WO 2008/042973.

Transfersomes, yet another type of liposomes, are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as those described herein, particularlay in emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The RNAi agent for use in the methods of the disclosure can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

Lipid Particles

RNAi agents, e.g., dsRNAs of in the disclosure may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; United States Patent publication No. 2010/0324120 and WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the disclosure.

Certain specific LNP formulations for delivery of RNAi agents have been described in the art, including, e.g., “LNP01” formulations as described in, e.g., WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are identified in the table below.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH- 50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in WO 2009/127060, which is hereby incorporated by reference. XTC comprising formulations are described in WO 2010/088537, the entire contents of which are hereby incorporated herein by reference. MC3 comprising formulations are described, e.g., in United States Patent Publication No. 2010/0324120, the entire contents of which are hereby incorporated by reference. ALNY-100 comprising formulations are described in WO 2010/054406, the entire contents of which are hereby incorporated herein by reference. C12-200 comprising formulations are described in WO 2010/129709, the entire contents of which are hereby incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. 2003/0027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the brain when treating APP-associated diseases or disorders.

The pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

Additional Formulations

i. Emulsions

The compositions of the present disclosure can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present disclosure, the compositions of RNAi agents and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used, and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or RNAi agents. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of RNAi agents and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of RNAi agents and nucleic acids.

Microemulsions of the present disclosure can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the RNAi agents and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An RNAi agent of the disclosure may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly RNAi agents, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of RNAi agents through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of RNAi agents through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rd., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of RNAi agents through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of RNAi agents at the cellular level can also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more RNAi agents and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a TMPRSS2-associated disorder. Examples of such agents include, but are not limited to an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e g , for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀ Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the disclosure lies generally within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the RNAi agents featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by nucleotide repeat expression. In any event, the administering physician can adjust the amount and timing of RNAi agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device, such as a device suitable for pulmonary administration, e.g., a device suitable for oral inhalative administration including nebulizers, metered-dose inhalers, and dry powder inhalers.

VIII. Methods for Inhibiting TMPRSS2 Expression

The present disclosure also provides methods of inhibiting expression of a TMPRSS2 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of a TMPRSS2 gene in the cell, thereby inhibiting expression of TMPRSS2 in the cell. In certain embodiments of the disclosure, expression of a TMPRSS2 gene is inhibited preferentially in the pulmonary system (e.g., lung, bronchial, alveoli) cells. In other embodiments of the disclosure, expression of a TMPRSS2 gene is inhibited preferentially in the liver (e.g., hepatocytes). In certain embodiments of the disclosure, expression of a TMPRSS2 gene is inhibited in the pulmonary system (e.g., lung, bronchial, alveoli) cells and in liver (e.g., hepatocytes) cells.

Contacting of a cell with a RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.

Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via Lipofectamine™-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., preferably 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art.

The phrase “inhibiting expression of a TMPRSS2 gene” or “inhibiting expression of TMPRSS2,” as used herein, includes inhibition of expression of any TMPRSS2 gene (such as, e.g., a mouse TMPRSS2 gene, a rat TMPRSS2 gene, a monkey TMPRSS2 gene, or a human TMPRSS2 gene) as well as variants or mutants of a TMPRSS2 gene that encode a TMPRSS2 protein. Thus, the TMPRSS2 gene may be a wild-type TMPRSS2 gene , a mutant TMPRSS2 gene , or a transgenic TMPRSS2 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a TMPRSS2 gene” includes any level of inhibition of a TMPRSS2 gene, e.g., at least partial suppression of the expression of a TMPRSS2 gene, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, preferably at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method. In a preferred method, inhibition is measured at a 10 nM concentration of the siRNA using the luciferase assay provided in Example 1.

The expression of a TMPRSS2 gene may be assessed based on the level of any variable associated with TMPRSS2 gene expression, e.g., TMPRSS2 mRNA level or TMPRSS2 protein level.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the disclosure, expression of a TMPRSS2 gene is inhibited by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of TMPRSS2, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a TMPRSS2 gene.

Inhibition of the expression of a TMPRSS2 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a TMPRSS2 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of a TMPRSS2 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a RNAi agent targeted to the genome of interest). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a TMPRSS2 gene may be assessed in terms of a reduction of a parameter that is functionally linked to a TMPRSS2 gene expression, e.g., TMPRSS2 protein expression, S protein priming, efficiency of viral entry, viral load. TMPRSS2 gene silencing may be determined in any cell expressing a TMPRSS2 gene, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of a TMPRSS2 protein may be manifested by a reduction in the level of the TMPRSS2 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of genome suppression, the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of a TMPRSS2 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of TMPRSS2 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of TMPRSS2 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the TMPRSS2 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating TMPRSS2 mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of TMPRSS2 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific TMPRSS2 nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to TMPRSS2 RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known RNA detection methods for use in determining the level of TMPRSS2 mRNA.

An alternative method for determining the level of expression of TMPRSS2 in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of TMPRSS2 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan^(Tm) System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of TMPRSS2 expression or mRNA level.

The expression level of TMPRSS2 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of TMPRSS2 expression level may also comprise using nucleic acid probes in solution.

In some embodiments, the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of TMPRSS2 nucleic acids.

The level of TMPRSS2 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of TMPRSS2 proteins.

In some embodiments, the efficacy of the methods of the disclosure in the treatment of a TMPRSS2-related disease is assessed by a decrease in TMPRSS2 mRNA level (e.g, by assessment of a TMPRSS2 level, e.g., in the lung, by biopsy, or otherwise).

In some embodiments, the efficacy of the methods of the disclosure in the treatment of a TMPRSS2-related disease is assessed by a decrease in TMPRSS2 mRNA level (e.g, by assessment of a liver sample for TMPRSS2 level, by biopsy, or otherwise).

In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of TMPRSS2 may be assessed using measurements of the level or change in the level of TMPRSS2 mRNA or TMPRSS2 protein in a sample derived from a specific site within the subject, e.g., lung and/or liver cells. In certain embodiments, the methods include a clinically relevant inhibition of expression of TMPRSS2, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of TMPRSS2.

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

IX. Methods of Treating or Preventing TMPRSS2-Associated Diseases

The present disclosure also provides methods of using a RNAi agent of the disclosure or a composition containing a RNAi agent of the disclosure to reduce or inhibit TMPRSS2 expression in a cell. The methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcripts of a TMPRSS2 gene, thereby inhibiting expression of the TMPRSS2 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of TMPRSS2 may be determined by determining the mRNA expression level of a TMPRSS2 gene using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a TMPRSS2 protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques.

In the methods of the disclosure the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a TMPRSS2 gene. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human lung cell. In one embodiment, the cell is a human cell, e.g., a human liver cell. In one embodiment, the cell is a human cell, e.g., a human lung cell and a human liver cell.

TMPRSS2 expression is inhibited in the cell by at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or about 100%, i.e., to below the level of detection. In preferred embodiments, TMPRSS2 expression is inhibited by at least 50%.

The in vivo methods of the disclosure may include administering to a subject a composition containing a RNAi agent, where the RNAi agent includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the TMPRSS2 gene of the subject to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by pulmonary delivery, e.g., oral inhalation or intranasal delivery.

In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of TMPRSS2, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration or oral inhalative administration. Pulmonary system administration may be via a syringe, a dropper, atomization, or use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI) device.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present disclosure also provides methods for inhibiting the expression of a TMPRSS2 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a TMPRSS2 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the RNA transcript of the TMPRSS2 gene, thereby inhibiting expression of the TMPRSS2 gene in the cell. Reduction in genome expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a lung biopsy sample serves as the tissue material for monitoring the reduction in TMPRSS2 gene or protein expression (or of a proxy therefore).

The present disclosure further provides methods of treatment of a subject in need thereof. The treatment methods of the disclosure include administering an RNAi agent of the disclosure to a subject, e.g., a subject that would benefit from inhibition of TMPRSS2 expression, in a therapeutically effective amount of a RNAi agent targeting a TMPRSS2 gene or a pharmaceutical composition comprising a RNAi agent targeting a TMPRSS2 gene.

In addition, the present disclosure provides methods of preventing, treating or inhibiting the progression of a TMPRSS2-associated disease or disorder, e.g., a coronavirus-associated disease, such as severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome-2 (SARS-2).

The methods include administering to the subject a therapeutically effective amount of any of the RNAi agent, e.g., dsRNA agents, or the pharmaceutical composition provided herein, thereby preventing, treating, or inhibiting the progression of the TMPRSS2-associated disease or disorder in the subject, such as COVID-19.

An RNAi agent of the disclosure may be administered as a “free RNAi agent.” A free RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject.

Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of TMPRSS2 gene expression are those having a TMPRSS2-associated disease, subjects at risk of developing a TMPRSS2-associate disease, e.g., subjects of an age greater than 60 years and/or subjects who are immunocompromised, and subjects at risk of developing a TMPRSS2-associate disease, e.g., during an epidemic or pandemic.

The disclosure further provides methods for the use of a RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of TMPRSS2 expression, e.g., a subject having a TMPRSS2-associated disorder, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an RNAi agent targeting TMPRSS2 is administered in combination with, e.g., an agent useful in treating a TMPRSS2-associated disorder as described elsewhere herein or as otherwise known in the art. For example, additional agents and treatments suitable for treating a subject that would benefit from reducton in TMPRSS2 expression, e.g., a subject having a TMPRSS2-associated disorder, may include agents currently used to treat symptoms of TMPRSS2-associated disorder. The RNAi agent and additional therapeutic agents may be administered at the same time or in the same combination, e.g., via pulmonary system administration, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

Exemplary additional therapeutics and treatments include, for example, an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.

In one embodiment, the method includes administering a composition featured herein such that expression of the target TMPRSS2 gene is decreased, for at least one month. In preferred embodiments, expression is decreased for at least 2 months, or 6 months.

Preferably, the RNAi agents useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target TMPRSS2 gene. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the disclosure may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a TMPRSS2-associated disorder. By “reduction” in this context is meant a statistically significant or clinically significant decrease in such level. The reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a RNAi agent targeting TMPRSS2 or pharmaceutical composition thereof, “effective against” a TMPRSS2-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating TMPRSS2-associated disorders and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50%, or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using a RNAi agent or RNAi agent formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The RNAi agent can be administered via the pulmonaty sysyem over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the RNAi agent can reduce TMPRSS2 levels, e.g., in a cell, tissue, blood, lung sample or other compartment of the patient by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70,% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In a preferred embodiment, administration of the RNAi agent can reduce TMPRSS2 levels, e.g., in a cell, tissue, blood, pulmonary system sample or other compartment of the patient by at least 50%.

Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the RNAi agent can be administered by pulmonary administration or subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired, e.g., monthly dose of RNAi agent to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimine may include administration of a therapeutic amount of RNAi agent on a regular basis, such as monthly or extending to once a quarter, twice per year, once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RNAi agents and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

An inforrmal Sequence Listing is filed herewith and forms part of the specification as filed.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, and the Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES Example 1 iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Design

The selection of siRNA designs targeting human transmembrane serine protease (TMPRSS2) gene (human NCBI refseqID: NM_005656.4; NCBI GenelD: 7113) were designed using custo R and Python scripts. The human NM_005656.4 REFSEQ mRNA has a length of 3450 bases.

A detailed list of a set of the unmodified siRNA sense and antisense strand sequences targeting TMPRSS2 is shown in Table 2.

A detailed list of a set of the modified siRNA sense and antisense strand sequences targeting TMPRSS2 is shown in Table 3.

It is to be understood that, throughout the application, a duplex name without a decimal is equivalent to a duplex name with a decimal which merely references the batch number of the duplex. For example, AD-1230521 is equivalent to AD-1230521.

siRNA Synthesis

siRNAs were synthesized and annealed using routine methods known in the art. Briefly, siRNA sequences were synthesized on a 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. The solid support was controlled pore glass (500-1000 Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universal solid support (AM Chemicals), or the first nucleotide of interest. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, Wis.), Hongene (China), or Chemgenes (Wilmington, Mass., USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs. Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”).

Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 μL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2′-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 μL of dimethyl sulfoxide (DMSO) and 300 μL TEA.3HF and the solution was incubated for approximately 30 mins at 60 ° C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively.

Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 μM in 1× PBS in 96 well plates, the plate sealed, incubated at 100 ° C. for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.

Example 2 In Vitro Screening of siRNA Duplexes Cell Culture and Transfections

Cells, e.g., pulmonary system cells, are cultured according to standard methods and are transfected with the iRNA duplex of interest. For example, primary human hepatocytes (PHH) were transfected by adding 7.5 μL of Opti-MEM plus 0.1 μL of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 2.5 μL of each siRNA duplex to an individual well in a 384-well plate. The cells were then incubated at room temperature for 15 minutes. Forty μL of MEDIA containing −1.5×10⁴ cells was then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose xperiments were performed at 10 nM, 1 nM, and 0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 3 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

cDNA synthesis

For cDNA synthesis, a master mix of 1.5 μl 10×Buffer, 0.6 μl 10×dNTPs, 1.5μ1 Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNase inhibitor and 9.9 μl of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

Real Time PCR

Two microlitre (μl) of cDNA were added to a master mix containing 0.5 μl of human GAPDH TaqMan Probe (4326317E), 0.5 μl human APOC3, 2 μl nuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche).

To calculate relative fold change, data were analyzed using the AACt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or mock-transfected. The sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO:15) and antisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO:16).

The results of the screening of the dsRNA agents listed in Tables 3 and 5 in primary human hepatocytes (PHH) are shown in Table 4.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′- phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine -3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide, modified or unmodified a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L10 N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol) L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3)

Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose) Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Agn) Adenosine-glycol nucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn) Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphonate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine-3′-phosphate dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine dUs 2′-deoxyuridine-3′-phosphorothioate (C2p) cytidine-2′-phosphate (G2p) guanosine-2′-phosphate (U2p) uridine-2′-phosphate (A2p) adenosine-2′-phosphate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate

TABLE 2 Unmodified Sense and Antisense Strand TMPRSS2 dsRNA Sequences SEQ SEQ Duplex ID Range in ID Range in Name Sense Sequence 5′ to 3′ NO: NM_005656.4 Antisense Sequence 5′ to 3′ NO: NM_005656.4 AD-1230521 UUCAUUUAACUCUUUGAAACA 17 2580-2600 UGUUUCAAAGAGUUAAAUGAAGG 317 2578-2600 AD-1230522 AAACGUCUUCCUUCUUUAUUA 18 3065-3085 UAAUAAAGAAGGAAGACGUUUUC 318 3063-3085 AD-1230523 AACGUCUUCCUUCUUUAUUGA 19 3066-3086 UCAAUAAAGAAGGAAGACGUUUU 319 3064-3086 AD-1230524 UGGUGAAAACGUCUUCCUUCA 20 3059-3079 UGAAGGAAGACGUUUUCACCAUU 320 3057-3079 AD-1230525 UGGUUUCUUUACGCUGUAUAA 21 841-861 UUAUACAGCGUAAAGAAACCACU 321 839-861 AD-1230526 UUCCAGAUACCUAUCAUUACA 22  92-112 UGUAAUGAUAGGUAUCUGGAAUG 322  90-112 AD-1230527 UCCAGAUACCUAUCAUUACUA 23  93-113 UAGUAAUGAUAGGUAUCUGGAAU 323  91-113 AD-1230528 CAGAUACCUAUCAUUACUCGA 24  95-115 UCGAGUAAUGAUAGGUAUCUGGA 324  93-115 AD-1230529 GAUACCUAUCAUUACUCGAUA 25  97-117 UAUCGAGUAAUGAUAGGUAUCUG 325  95-117 AD-1230530 CGUGGAAAAACCUCUUAACAA 26 1025-1045 UUGUUAAGAGGUUUUUCCACGCA 326 1023-1045 AD-1230531 AGUGAUUUCUCAUCCAAAUUA 27 1124-1144 UAAUUUGGAUGAGAAAUCACUUU 327 1122-1144 AD-1230532 UCUCAUCCAAAUUAUGACUCA 28 1131-1151 UGAGUCAUAAUUUGGAUGAGAAA 328 1129-1151 AD-1230533 AUCCAAAUUAUGACUCCAAGA 29 1135-1155 UCUUGGAGUCAUAAUUUGGAUGA 329 1133-1155 AD-1230534 ACUGGAUUUAUCGACAAAUGA 30 1579-1599 UCAUUUGUCGAUAAAUCCAGUCC 330 1577-1599 AD-1230535 UUUGAUGUCUCCAAGUAGUCA 31 2556-2576 UGACUACUUGGAGACAUCAAAAG 331 2554-2576 AD-1230536 CAAGACACAUCCUAAAAGGUA 32 3032-3052 UACCUUUUAGGAUGUGUCUUGGG 332 3030-3052 AD-1230537 GUAAUGGUGAAAACGUCUUCA 33 3055-3075 UGAAGACGUUUUCACCAUUACAA 333 3053-3075 AD-1230538 UGAAAACGUCUUCCUUCUUUA 34 3062-3082 UAAAGAAGGAAGACGUUUUCACC 334 3060-3082 AD-1230539 GAAAACGUCUUCCUUCUUUAA 35 3063-3083 UUAAAGAAGGAAGACGUUUUCAC 335 3061-3083 AD-1230540 AACUGUAAAGUUCAAUUGUGA 36 3137-3157 UCACAAUUGAACUUUACAGUUUA 336 3135-3157 AD-1230541 GUACUCAUCUCAGAGGAAGUA 37 614-634 UACUUCCUCUGAGAUGAGUACAC 337 612-634 AD-1230542 CGGCAAUGUCGAUAUCUAUAA 38 782-802 UUAUAGAUAUCGACAUUGCCGGC 338 780-802 AD-1230543 GGCAAUGUCGAUAUCUAUAAA 39 783-803 UUUAUAGAUAUCGACAUUGCCGG 339 781-803 AD-1230544 GCAAUGUCGAUAUCUAUAAAA 40 784-804 UUUUAUAGAUAUCGACAUUGCCG 340 782-804 AD-1230545 CAAUGUCGAUAUCUAUAAAAA 41 785-805 UUUUUAUAGAUAUCGACAUUGCC 341 783-805 AD-1230546 GUGGUUUCUUUACGCUGUAUA 42 840-860 UAUACAGCGUAAAGAAACCACUG 342 838-860 AD-1230547 AUUCCAGAUACCUAUCAUUAA 43  91-111 UUAAUGAUAGGUAUCUGGAAUGU 343  89-111 AD-1230548 CCAGAUACCUAUCAUUACUCA 44  94-114 UGAGUAAUGAUAGGUAUCUGGAA 344  92-114 AD-1230549 AGAUACCUAUCAUUACUCGAA 45  96-116 UUCGAGUAAUGAUAGGUAUCUGG 345  94-116 AD-1230550 GUGAUUUCUCAUCCAAAUUAA 46 1125-1145 UUAAUUUGGAUGAGAAAUCACUU 346 1123-1145 AD-1230551 UGAUUUCUCAUCCAAAUUAUA 47 1126-1146 UAUAAUUUGGAUGAGAAAUCACU 347 1124-1146 AD-1230552 AUUUCUCAUCCAAAUUAUGAA 48 1128-1148 UUCAUAAUUUGGAUGAGAAAUCA 348 1126-1148 AD-1230553 UUUCUCAUCCAAAUUAUGACA 49 1129-1149 UGUCAUAAUUUGGAUGAGAAAUC 349 1127-1149 AD-1230554 UUCUCAUCCAAAUUAUGACUA 50 1130-1150 UAGUCAUAAUUUGGAUGAGAAAU 350 1128-1150 AD-1230555 AGGUCACUUCAUUUUUAUUAA 51 1708-1728 UUAAUAAAAAUGAAGUGACCUCU 351 1706-1728 AD-1230556 AUGUUUCUACACAUUGCUACA 52 2512-2532 UGUAGCAAUGUGUAGAAACAUAA 352 2510-2532 AD-1230557 UCAUUUAACUCUUUGAAACUA 53 2581-2601 UAGUUUCAAAGAGUUAAAUGAAG 353 2579-2601 AD-1230558 CUAGGACUUAACCUUGAAAUA 54 2903-2923 UAUUUCAAGGUUAAGUCCUAGCU 354 2901-2923 AD-1230559 AGACACAUCCUAAAAGGUGUA 55 3034-3054 UACACCUUUUAGGAUGUGUCUUG 355 3032-3054 AD-1230560 GACACAUCCUAAAAGGUGUUA 56 3035-3055 UAACACCUUUUAGGAUGUGUCUU 356 3033-3055 AD-1230561 AUGGUGAAAACGUCUUCCUUA 57 3058-3078 UAAGGAAGACGUUUUCACCAUUA 357 3056-3078 AD-1230562 GUGAAAACGUCUUCCUUCUUA 58 3061-3081 UAAGAAGGAAGACGUUUUCACCA 358 3059-3081 AD-1230563 AAAACGUCUUCCUUCUUUAUA 59 3064-3084 UAUAAAGAAGGAAGACGUUUUCA 359 3062-3084 AD-1230564 AUGCCUGUUCUUCAAAAGCAA 60 820-840 UUGCUUUUGAAGAACAGGCAUCA 360 818-840 AD-1230565 GCCUGUUCUUCAAAAGCAGUA 61 822-842 UACUGCUUUUGAAGAACAGGCAU 361 820-842 AD-1230566 UUCAAAAGCAGUGGUUUCUUA 62 830-850 UAAGAAACCACUGCUUUUGAAGA 362 828-850 AD-1230567 CUAUCAUUACUCGAUGCUGUA 63 102-122 UACAGCAUCGAGUAAUGAUAGGU 363 100-122 AD-1230568 UGCGUGGAAAAACCUCUUAAA 64 1023-1043 UUUAAGAGGUUUUUCCACGCAGU 364 1021-1043 AD-1230569 CAAGUAGAAAAAGUGAUUUCA 65 1113-1133 UGAAAUCACUUUUUCUACUUGGU 365 1111-1133 AD-1230570 AAGUAGAAAAAGUGAUUUCUA 66 1114-1134 UAGAAAUCACUUUUUCUACUUGG 366 1112-1134 AD-1230571 GUAGAAAAAGUGAUUUCUCAA 67 1116-1136 UUGAGAAAUCACUUUUUCUACUU 367 1114-1136 AD-1230572 UAGAAAAAGUGAUUUCUCAUA 68 1117-1137 UAUGAGAAAUCACUUUUUCUACU 368 1115-1137 AD-1230573 AAAAGUGAUUUCUCAUCCAAA 69 1121-1141 UUUGGAUGAGAAAUCACUUUUUC 369 1119-1141 AD-1230574 GAUUUCUCAUCCAAAUUAUGA 70 1127-1147 UCAUAAUUUGGAUGAGAAAUCAC 370 1125-1147 AD-1230575 AAGCCUCUGACUUUCAACGAA 71 1191-1211 UUCGUUGAAAGUCAGAGGCUUCU 371 1189-1211 AD-1230576 GCUAGUCACUGGAAAUUGAGA 72 2440-2460 UCUCAAUUUCCAGUGACUAGCAG 372 2438-2460 AD-1230577 UUUUGAUGUCUCCAAGUAGUA 73 2555-2575 UACUACUUGGAGACAUCAAAAGC 373 2553-2575 AD-1230578 GAUGUGUUUUGUUUUGGACUA 74 2731-2751 UAGUCCAAAACAAAACACAUCUU 374 2729-2751 AD-1230579 UUUUGUUUUGGACUCUCUGUA 75 2737-2757 UACAGAGAGUCCAAAACAAAACA 375 2735-2757 AD-1230580 GCUGGUUUGCAAGAAUGAAAA 76 2868-2888 UUUUCAUUCUUGCAAACCAGCCU 376 2866-2888 AD-1230581 UGGUUUGCAAGAAUGAAAUGA 77 2870-2890 UCAUUUCAUUCUUGCAAACCAGC 377 2868-2890 AD-1230582 GGUUUGCAAGAAUGAAAUGAA 78 2871-2891 UUCAUUUCAUUCUUGCAAACCAG 378 2869-2891 AD-1230583 UAGGACUUAACCUUGAAAUGA 79 2904-2924 UCAUUUCAAGGUUAAGUCCUAGC 379 2902-2924 AD-1230584 GGUGAAAACGUCUUCCUUCUA 80 3060-3080 UAGAAGGAAGACGUUUUCACCAU 380 3058-3080 AD-1230585 ACGUCUUCCUUCUUUAUUGCA 81 3067-3087 UGCAAUAAAGAAGGAAGACGUUU 381 3065-3087 AD-1230586 CGUCUUCCUUCUUUAUUGCCA 82 3068-3088 UGGCAAUAAAGAAGGAAGACGUU 382 3066-3088 AD-1230587 CUGUAAAGUUCAAUUGUGAAA 83 3139-3159 UUUCACAAUUGAACUUUACAGUU 383 3137-3159 AD-1230588 GCACCUCAAAGACUAAGAAAA 84 364-384 UUUUCUUAGUCUUUGAGGUGCAC 384 362-384 AD-1230589 CACCUCAAAGACUAAGAAAGA 85 365-385 UCUUUCUUAGUCUUUGAGGUGCA 385 363-385 AD-1230590 AUCCACCAGCUUUAUGAAACA 86 749-769 UGUUUCAUAAAGCUGGUGGAUCC 386 747-769 AD-1230591 UGAUGCCUGUUCUUCAAAAGA 87 818-838 UCUUUUGAAGAACAGGCAUCACU 387 816-838 AD-1230592 GAUGCCUGUUCUUCAAAAGCA 88 819-839 UGCUUUUGAAGAACAGGCAUCAC 388 817-839 AD-1230593 UGCCUGUUCUUCAAAAGCAGA 89 821-841 UCUGCUUUUGAAGAACAGGCAUC 389 819-841 AD-1230594 UCUUCAAAAGCAGUGGUUUCA 90 828-848 UGAAACCACUGCUUUUGAAGAAC 390 826-848 AD-1230595 CAUUCCAGAUACCUAUCAUUA 91  90-110 UAAUGAUAGGUAUCUGGAAUGUU 391  88-110 AD-1230596 ACCUAUCAUUACUCGAUGCUA 92 100-120 UAGCAUCGAGUAAUGAUAGGUAU 392  98-120 AD-1230597 GCGUGGAAAAACCUCUUAACA 93 1024-1044 UGUUAAGAGGUUUUUCCACGCAG 393 1022-1044 AD-1230598 CAUUACUCGAUGCUGUUGAUA 94 106-126 UAUCAACAGCAUCGAGUAAUGAU 394 104-126 AD-1230599 UUACUCGAUGCUGUUGAUAAA 95 108-128 UUUAUCAACAGCAUCGAGUAAUG 395 106-128 AD-1230600 AAGAACAAUGACAUUGCGCUA 96 1158-1178 UAGCGCAAUGUCAUUGUUCUUGG 396 1156-1178 AD-1230601 ACAGCAAGAUGGCUUUGAACA 97 127-147 UGUUCAAAGCCAUCUUGCUGUUA 397 125-147 AD-1230602 CAGCAAGAUGGCUUUGAACUA 98 128-148 UAGUUCAAAGCCAUCUUGCUGUU 398 126-148 AD-1230603 UCAGAGGUCACUUCAUUUUUA 99 1704-1724 UAAAAAUGAAGUGACCUCUGAAU 399 1702-1724 AD-1230604 CAGAGGUCACUUCAUUUUUAA 100 1705-1725 UUAAAAAUGAAGUGACCUCUGAA 400 1703-1725 AD-1230605 UGUUAUGUUUCUACACAUUGA 101 2508-2528 UCAAUGUGUAGAAACAUAACAUG 401 2506-2528 AD-1230606 UAUGUUUCUACACAUUGCUAA 102 2511-2531 UUAGCAAUGUGUAGAAACAUAAC 402 2509-2531 AD-1230607 ACGUUCUAUAAAUGAAUGUGA 103 2672-2692 UCACAUUCAUUUAUAGAACGUUA 403 2670-2692 AD-1230608 GUUUUGUUUUGGACUCUCUGA 104 2736-2756 UCAGAGAGUCCAAAACAAAACAC 404 2734-2756 AD-1230609 GUUUGCAAGAAUGAAAUGAAA 105 2872-2892 UUUCAUUUCAUUCUUGCAAACCA 405 2870-2892 AD-1230610 AAGAAUGAAAUGAAUGAUUCA 106 2878-2898 UGAAUCAUUCAUUUCAUUCUUGC 406 2876-2898 AD-1230611 GCUAGGACUUAACCUUGAAAA 107 2902-2922 UUUUCAAGGUUAAGUCCUAGCUG 407 2900-2922 AD-1230612 AAGACACAUCCUAAAAGGUGA 108 3033-3053 UCACCUUUUAGGAUGUGUCUUGG 408 3031-3053 AD-1230613 UGUAAUGGUGAAAACGUCUUA 109 3054-3074 UAAGACGUUUUCACCAUUACAAC 409 3052-3074 AD-1230614 AAUGGUGAAAACGUCUUCCUA 110 3057-3077 UAGGAAGACGUUUUCACCAUUAC 410 3055-3077 AD-1230615 ACUGUAAAGUUCAAUUGUGAA 111 3138-3158 UUCACAAUUGAACUUUACAGUUU 411 3136-3158 AD-1230616 UGUAAAGUUCAAUUGUGAAAA 112 3140-3160 UUUUCACAAUUGAACUUUACAGU 412 3138-3160 AD-1230617 ACCUCAAAGACUAAGAAAGCA 113 366-386 UGCUUUCUUAGUCUUUGAGGUGC 413 364-386 AD-1230618 CCUCAAAGACUAAGAAAGCAA 114 367-387 UUGCUUUCUUAGUCUUUGAGGUG 414 365-387 AD-1230619 CUCAAAGACUAAGAAAGCACA 115 368-388 UGUGCUUUCUUAGUCUUUGAGGU 415 366-388 AD-1230620 CACCAGCUUUAUGAAACUGAA 116 752-772 UUCAGUUUCAUAAAGCUGGUGGA 416 750-772 AD-1230621 CCGGCAAUGUCGAUAUCUAUA 117 781-801 UAUAGAUAUCGACAUUGCCGGCA 417 779-801 AD-1230622 GUUCUUCAAAAGCAGUGGUUA 118 826-846 UAACCACUGCUUUUGAAGAACAG 418 824-846 AD-1230623 CUUCAAAAGCAGUGGUUUCUA 119 829-849 UAGAAACCACUGCUUUUGAAGAA 419 827-849 AD-1230624 UUGAACAUUCCAGAUACCUAA 120  85-105 UUAGGUAUCUGGAAUGUUCAAUA 420  83-105 AD-1230625 ACAUUCCAGAUACCUAUCAUA 121  89-109 UAUGAUAGGUAUCUGGAAUGUUC 421  87-109 AD-1230626 UCAUUACUCGAUGCUGUUGAA 122 105-125 UUCAACAGCAUCGAGUAAUGAUA 422 103-125 AD-1230627 AGUAGAAAAAGUGAUUUCUCA 123 1115-1135 UGAGAAAUCACUUUUUCUACUUG 423 1113-1135 AD-1230628 CUCCAAGACCAAGAACAAUGA 124 1148-1168 UCAUUGUUCUUGGUCUUGGAGUC 424 1146-1168 AD-1230629 CAAGACCAAGAACAAUGACAA 125 1151-1171 UUGUCAUUGUUCUUGGUCUUGGA 425 1149-1171 AD-1230630 CAAGAUGGCUUUGAACUCAGA 126 131-151 UCUGAGUUCAAAGCCAUCUUGCU 426 129-151 AD-1230631 GACGUGGUAGUCACUUGUAAA 127 2161-2181 UUUACAAGUGACUACCACGUCAC 427 2159-2181 AD-1230632 GUUAUGUUUCUACACAUUGCA 128 2509-2529 UGCAAUGUGUAGAAACAUAACAU 428 2507-2529 AD-1230633 UUUGCAAGAAUGAAAUGAAUA 129 2873-2893 UAUUCAUUUCAUUCUUGCAAACC 429 2871-2893 AD-1230634 AGGACUUAACCUUGAAAUGGA 130 2905-2925 UCCAUUUCAAGGUUAAGUCCUAG 430 2903-2925 AD-1230635 UGUGAAAAUGAAUAUCAUGCA 131 3153-3173 UGCAUGAUAUUCAUUUUCACAAU 431 3151-3173 AD-1230636 AGUGAUGCCUGUUCUUCAAAA 132 816-836 UUUUGAAGAACAGGCAUCACUGU 432 814-836 AD-1230637 UUCUUCAAAAGCAGUGGUUUA 133 827-847 UAAACCACUGCUUUUGAAGAACA 433 825-847 AD-1230638 UCAAAAGCAGUGGUUUCUUUA 134 831-851 UAAAGAAACCACUGCUUUUGAAG 434 829-851 AD-1230639 CAAAAGCAGUGGUUUCUUUAA 135 832-852 UUAAAGAAACCACUGCUUUUGAA 435 830-852 AD-1230640 GGUUUCUUUACGCUGUAUAGA 136 842-862 UCUAUACAGCGUAAAGAAACCAC 436 840-862 AD-1230641 AUACCUAUCAUUACUCGAUGA 137  98-118 UCAUCGAGUAAUGAUAGGUAUCU 437  96-118 AD-1230642 AUCAUUACUCGAUGCUGUUGA 138 104-124 UCAACAGCAUCGAGUAAUGAUAG 438 102-124 AD-1230643 AAAAAGUGAUUUCUCAUCCAA 139 1120-1140 UUGGAUGAGAAAUCACUUUUUCU 439 1118-1140 AD-1230644 ACUCCAAGACCAAGAACAAUA 140 1147-1167 UAUUGUUCUUGGUCUUGGAGUCA 440 1145-1167 AD-1230645 CCAAGACCAAGAACAAUGACA 141 1150-1170 UGUCAUUGUUCUUGGUCUUGGAG 441 1148-1170 AD-1230646 AAGACCAAGAACAAUGACAUA 142 1152-1172 UAUGUCAUUGUUCUUGGUCUUGG 442 1150-1172 AD-1230647 CUGGAUUUAUCGACAAAUGAA 143 1580-1600 UUCAUUUGUCGAUAAAUCCAGUC 443 1578-1600 AD-1230648 UUCAGAGGUCACUUCAUUUUA 144 1703-1723 UAAAAUGAAGUGACCUCUGAAUC 444 1701-1723 AD-1230649 AGAGGUCACUUCAUUUUUAUA 145 1706-1726 UAUAAAAAUGAAGUGACCUCUGA 445 1704-1726 AD-1230650 GAGGUCACUUCAUUUUUAUUA 146 1707-1727 UAAUAAAAAUGAAGUGACCUCUG 446 1705-1727 AD-1230651 GGAACAGAAACAUUUUUGUUA 147 2183-2203 UAACAAAAAUGUUUCUGUUCCCC 447 2181-2203 AD-1230652 UGCUAGUCACUGGAAAUUGAA 148 2439-2459 UUCAAUUUCCAGUGACUAGCAGG 448 2437-2459 AD-1230653 UUAUGUUUCUACACAUUGCUA 149 2510-2530 UAGCAAUGUGUAGAAACAUAACA 449 2508-2530 AD-1230654 CUUUUGAUGUCUCCAAGUAGA 150 2554-2574 UCUACUUGGAGACAUCAAAAGCU 450 2552-2574 AD-1230655 UGAUGUCUCCAAGUAGUCCAA 151 2558-2578 UUGGACUACUUGGAGACAUCAAA 451 2556-2578 AD-1230656 UUCUAUAAAUGAAUGUGCUGA 152 2675-2695 UCAGCACAUUCAUUUAUAGAACG 452 2673-2695 AD-1230657 AGAAUGAAAUGAAUGAUUCUA 153 2879-2899 UAGAAUCAUUCAUUUCAUUCUUG 453 2877-2899 AD-1230658 AAUGAAAUGAAUGAUUCUACA 154 2881-2901 UGUAGAAUCAUUCAUUUCAUUCU 454 2879-2901 AD-1230659 GGACUUAACCUUGAAAUGGAA 155 2906-2926 UUCCAUUUCAAGGUUAAGUCCUA 455 2904-2926 AD-1230660 GACUUAACCUUGAAAUGGAAA 156 2907-2927 UUUCCAUUUCAAGGUUAAGUCCU 456 2905-2927 AD-1230661 AGUCAUGCAAUCCCAUUUGCA 157 2927-2947 UGCAAAUGGGAUUGCAUGACUUU 457 2925-2947 AD-1230662 GUCAUGCAAUCCCAUUUGCAA 158 2928-2948 UUGCAAAUGGGAUUGCAUGACUU 458 2926-2948 AD-1230663 GUGCACCUCAAAGACUAAGAA 159 362-382 UUCUUAGUCUUUGAGGUGCACAC 459 360-382 AD-1230664 UCCACCAGCUUUAUGAAACUA 160 750-770 UAGUUUCAUAAAGCUGGUGGAUC 460 748-770 AD-1230665 AGCUUUAUGAAACUGAACACA 161 756-776 UGUGUUCAGUUUCAUAAAGCUGG 461 754-776 AD-1230666 AUGAAACUGAACACAAGUGCA 162 762-782 UGCACUUGUGUUCAGUUUCAUAA 462 760-782 AD-1230667 AUUGAACAUUCCAGAUACCUA 163  84-104 UAGGUAUCUGGAAUGUUCAAUAU 463  82-104 AD-1230668 AAAAGCAGUGGUUUCUUUACA 164 833-853 UGUAAAGAAACCACUGCUUUUGA 464 831-853 AD-1230669 GUUUCUUUACGCUGUAUAGCA 165 843-863 UGCUAUACAGCGUAAAGAAACCA 465 841-863 AD-1230670 AUUACUCGAUGCUGUUGAUAA 166 107-127 UUAUCAACAGCAUCGAGUAAUGA 466 105-127 AD-1230671 GACCAAGAACAAUGACAUUGA 167 1154-1174 UCAAUGUCAUUGUUCUUGGUCUU 467 1152-1174 AD-1230672 ACCAAGAACAAUGACAUUGCA 168 1155-1175 UGCAAUGUCAUUGUUCUUGGUCU 468 1153-1175 AD-1230673 AGCAAGAUGGCUUUGAACUCA 169 129-149 UGAGUUCAAAGCCAUCUUGCUGU 469 127-149 AD-1230674 CACGGACUGGAUUUAUCGACA 170 1574-1594 UGUCGAUAAAUCCAGUCCGUGAA 470 1572-1594 AD-1230675 ACGGACUGGAUUUAUCGACAA 171 1575-1595 UUGUCGAUAAAUCCAGUCCGUGA 471 1573-1595 AD-1230676 UGGAUUUAUCGACAAAUGAGA 172 1581-1601 UCUCAUUUGUCGAUAAAUCCAGU 472 1579-1601 AD-1230677 ACGUGGUAGUCACUUGUAAGA 173 2162-2182 UCUUACAAGUGACUACCACGUCA 473 2160-2182 AD-1230678 UAGUCACUGGAAAUUGAGGUA 174 2442-2462 UACCUCAAUUUCCAGUGACUAGC 474 2440-2462 AD-1230679 AAAUCAAGGAUGCUCAGUUUA 175 2471-2491 UAAACUGAGCAUCCUUGAUUUCC 475 2469-2491 AD-1230680 UCUAUAAAUGAAUGUGCUGAA 176 2676-2696 UUCAGCACAUUCAUUUAUAGAAC 476 2674-2696 AD-1230681 UAUAAAUGAAUGUGCUGAAGA 177 2678-2698 UCUUCAGCACAUUCAUUUAUAGA 477 2676-2698 AD-1230682 UUUGUUUUGGACUCUCUGUGA 178 2738-2758 UCACAGAGAGUCCAAAACAAAAC 478 2736-2758 AD-1230683 GGCUGGUUUGCAAGAAUGAAA 179 2867-2887 UUUCAUUCUUGCAAACCAGCCUG 479 2865-2887 AD-1230684 AGCUAGGACUUAACCUUGAAA 180 2901-2921 UUUCAAGGUUAAGUCCUAGCUGU 480 2899-2921 AD-1230685 UCCCAUUUGCAGGAUCUGUCA 181 2937-2957 UGACAGAUCCUGCAAAUGGGAUU 481 2935-2957 AD-1230686 AAAGUUCAAUUGUGAAAAUGA 182 3143-3163 UCAUUUUCACAAUUGAACUUUAC 482 3141-3163 AD-1230687 UGCACCUCAAAGACUAAGAAA 183 363-383 UUUCUUAGUCUUUGAGGUGCACA 483 361-383 AD-1230688 AAAGACUAAGAAAGCACUGUA 184 371-391 UACAGUGCUUUCUUAGUCUUUGA 484 369-391 AD-1230689 CAGGUGUACUCAUCUCAGAGA 185 609-629 UCUCUGAGAUGAGUACACCUGAA 485 607-629 AD-1230690 ACUCUAGCCAAGGAAUAGUGA 186 718-738 UCACUAUUCCUUGGCUAGAGUAA 486 716-738 AD-1230691 CAGUGAUGCCUGUUCUUCAAA 187 815-835 UUUGAAGAACAGGCAUCACUGUG 487 813-835 AD-1230692 AAAGCAGUGGUUUCUUUACGA 188 834-854 UCGUAAAGAAACCACUGCUUUUG 488 832-854 AD-1230693 GCAGUGGUUUCUUUACGCUGA 189 837-857 UCAGCGUAAAGAAACCACUGCUU 489 835-857 AD-1230694 CAGUGGUUUCUUUACGCUGUA 190 838-858 UACAGCGUAAAGAAACCACUGCU 490 836-858 AD-1230695 CCUAUCAUUACUCGAUGCUGA 191 101-121 UCAGCAUCGAGUAAUGAUAGGUA 491  99-121 AD-1230696 AUGACUCCAAGACCAAGAACA 192 1144-1164 UGUUCUUGGUCUUGGAGUCAUAA 492 1142-1164 AD-1230697 UCCAAGACCAAGAACAAUGAA 193 1149-1169 UUCAUUGUUCUUGGUCUUGGAGU 493 1147-1169 AD-1230698 UUCACGGACUGGAUUUAUCGA 194 1572-1592 UCGAUAAAUCCAGUCCGUGAAUA 494 1570-1592 AD-1230699 UGAUUCAGAGGUCACUUCAUA 195 1700-1720 UAUGAAGUGACCUCUGAAUCAUC 495 1698-1720 AD-1230700 GAUUCAGAGGUCACUUCAUUA 196 1701-1721 UAAUGAAGUGACCUCUGAAUCAU 496 1699-1721 AD-1230701 AUUCAGAGGUCACUUCAUUUA 197 1702-1722 UAAAUGAAGUGACCUCUGAAUCA 497 1700-1722 AD-1230702 AGUCACUGGAAAUUGAGGUCA 198 2443-2463 UGACCUCAAUUUCCAGUGACUAG 498 2441-2463 AD-1230703 GUUUCUACACAUUGCUACCUA 199 2514-2534 UAGGUAGCAAUGUGUAGAAACAU 499 2512-2534 AD-1230704 UUUCUACACAUUGCUACCUCA 200 2515-2535 UGAGGUAGCAAUGUGUAGAAACA 500 2513-2535 AD-1230705 UUGAUGUCUCCAAGUAGUCCA 201 2557-2577 UGGACUACUUGGAGACAUCAAAA 501 2555-2577 AD-1230706 GUUCUAUAAAUGAAUGUGCUA 202 2674-2694 UAGCACAUUCAUUUAUAGAACGU 502 2672-2694 AD-1230707 UGUUUUGGACUCUCUGUGGUA 203 2740-2760 UACCACAGAGAGUCCAAAACAAA 503 2738-2760 AD-1230708 UAACCAUGAGCACUACUCUAA 204 2821-2841 UUAGAGUAGUGCUCAUGGUUAUG 504 2819-2841 AD-1230709 GCAGGCUGGUUUGCAAGAAUA 205 2864-2884 UAUUCUUGCAAACCAGCCUGCUU 505 2862-2884 AD-1230710 CAAGAAUGAAAUGAAUGAUUA 206 2877-2897 UAAUCAUUCAUUUCAUUCUUGCA 506 2875-2897 AD-1230711 AAGACUAAGAAAGCACUGUGA 207 372-392 UCACAGUGCUUUCUUAGUCUUUG 507 370-392 AD-1230712 CUUCAGGUGUACUCAUCUCAA 208 606-626 UUGAGAUGAGUACACCUGAAGGA 508 604-626 AD-1230713 UUCAGGUGUACUCAUCUCAGA 209 607-627 UCUGAGAUGAGUACACCUGAAGG 509 605-627 AD-1230714 CCACCAGCUUUAUGAAACUGA 210 751-771 UCAGUUUCAUAAAGCUGGUGGAU 510 749-771 AD-1230715 AAGCAGUGGUUUCUUUACGCA 211 835-855 UGCGUAAAGAAACCACUGCUUUU 511 833-855 AD-1230716 AGCAGUGGUUUCUUUACGCUA 212 836-856 UAGCGUAAAGAAACCACUGCUUU 512 834-856 AD-1230717 CAAGAACAAUGACAUUGCGCA 213 1157-1177 UGCGCAAUGUCAUUGUUCUUGGU 513 1155-1177 AD-1230718 CGGACUGGAUUUAUCGACAAA 214 1576-1596 UUUGUCGAUAAAUCCAGUCCGUG 514 1574-1596 AD-1230719 GGAUUUAUCGACAAAUGAGGA 215 1582-1602 UCCUCAUUUGUCGAUAAAUCCAG 515 1580-1602 AD-1230720 UGACGUGGUAGUCACUUGUAA 216 2160-2180 UUACAAGUGACUACCACGUCACC 516 2158-2180 AD-1230721 CGUUCUAUAAAUGAAUGUGCA 217 2673-2693 UGCACAUUCAUUUAUAGAACGUU 517 2671-2693 AD-1230722 AGAAAGAUGUGUUUUGUUUUA 218 2726-2746 UAAAACAAAACACAUCUUUCUCU 518 2724-2746 AD-1230723 GAAAGAUGUGUUUUGUUUUGA 219 2727-2747 UCAAAACAAAACACAUCUUUCUC 519 2725-2747 AD-1230724 AGAUGUGUUUUGUUUUGGACA 220 2730-2750 UGUCCAAAACAAAACACAUCUUU 520 2728-2750 AD-1230725 UUGCAAGAAUGAAAUGAAUGA 221 2874-2894 UCAUUCAUUUCAUUCUUGCAAAC 521 2872-2894 AD-1230726 UGCAAGAAUGAAAUGAAUGAA 222 2875-2895 UUCAUUCAUUUCAUUCUUGCAAA 522 2873-2895 AD-1230727 GCAAGAAUGAAAUGAAUGAUA 223 2876-2896 UAUCAUUCAUUUCAUUCUUGCAA 523 2874-2896 AD-1230728 AAGUCAUGCAAUCCCAUUUGA 224 2926-2946 UCAAAUGGGAUUGCAUGACUUUC 524 2924-2946 AD-1230729 AUCCCAUUUGCAGGAUCUGUA 225 2936-2956 UACAGAUCCUGCAAAUGGGAUUG 525 2934-2956 AD-1230730 UCUGUAGAGAGCAGCAUUCCA 226 2969-2989 UGGAAUGCUGCUCUCUACAGAGG 526 2967-2989 AD-1230731 UAAUGGUGAAAACGUCUUCCA 227 3056-3076 UGGAAGACGUUUUCACCAUUACA 527 3054-3076 AD-1230732 GUGAAAAUGAAUAUCAUGCAA 228 3154-3174 UUGCAUGAUAUUCAUUUUCACAA 528 3152-3174 AD-1230733 UGAAAAUGAAUAUCAUGCAAA 229 3155-3175 UUUGCAUGAUAUUCAUUUUCACA 529 3153-3175 AD-1230734 CCUUCAGGUGUACUCAUCUCA 230 605-625 UGAGAUGAGUACACCUGAAGGAU 530 603-625 AD-1230735 CGACUGGAACGAGAACUACGA 231 656-676 UCGUAGUUCUCGUUCCAGUCGUC 531 654-676 AD-1230736 GGGACAUGGGCUAUAAGAAUA 232 691-711 UAUUCUUAUAGCCCAUGUCCCUG 532 689-711 AD-1230737 UUUUACUCUAGCCAAGGAAUA 233 714-734 UAUUCCUUGGCUAGAGUAAAAAU 533 712-734 AD-1230738 UACUCUAGCCAAGGAAUAGUA 234 717-737 UACUAUUCCUUGGCUAGAGUAAA 534 715-737 AD-1230739 UAUGAAACUGAACACAAGUGA 235 761-781 UCACUUGUGUUCAGUUUCAUAAA 535 759-781 AD-1230740 UACCUAUCAUUACUCGAUGCA 236  99-119 UGCAUCGAGUAAUGAUAGGUAUC 536  97-119 AD-1230741 AUUCACGGACUGGAUUUAUCA 237 1571-1591 UGAUAAAUCCAGUCCGUGAAUAC 537 1569-1591 AD-1230742 UCACGGACUGGAUUUAUCGAA 238 1573-1593 UUCGAUAAAUCCAGUCCGUGAAU 538 1571-1593 AD-1230743 AUGAAAACCAUGGAUACCAAA 239 178-198 UUUGGUAUCCAUGGUUUUCAUAG 539 176-198 AD-1230744 CGUGGUAGUCACUUGUAAGGA 240 2163-2183 UCCUUACAAGUGACUACCACGUC 540 2161-2183 AD-1230745 GCGAAGAAGAGAAAGAUGUGA 241 2717-2737 UCACAUCUUUCUCUUCUUCGCCG 541 2715-2737 AD-1230746 GAAGAAGAGAAAGAUGUGUUA 242 2719-2739 UAACACAUCUUUCUCUUCUUCGC 542 2717-2739 AD-1230747 AAGAAGAGAAAGAUGUGUUUA 243 2720-2740 UAAACACAUCUUUCUCUUCUUCG 543 2718-2740 AD-1230748 AGAAGAGAAAGAUGUGUUUUA 244 2721-2741 UAAAACACAUCUUUCUCUUCUUC 544 2719-2741 AD-1230749 AUAACCAUGAGCACUACUCUA 245 2820-2840 UAGAGUAGUGCUCAUGGUUAUGG 545 2818-2840 AD-1230750 CAGGCUGGUUUGCAAGAAUGA 246 2865-2885 UCAUUCUUGCAAACCAGCCUGCU 546 2863-2885 AD-1230751 UCAUGCAAUCCCAUUUGCAGA 247 2929-2949 UCUGCAAAUGGGAUUGCAUGACU 547 2927-2949 AD-1230752 CCCAUUUGCAGGAUCUGUCUA 248 2938-2958 UAGACAGAUCCUGCAAAUGGGAU 548 2936-2958 AD-1230753 CUCAUCUCAGAGGAAGUCCUA 249 617-637 UAGGACUUCCUCUGAGAUGAGUA 549 615-637 AD-1230754 GACGACUGGAACGAGAACUAA 250 654-674 UUAGUUCUCGUUCCAGUCGUCUU 550 652-674 AD-1230755 CUAGCCAAGGAAUAGUGGAUA 251 721-741 UAUCCACUAUUCCUUGGCUAGAG 551 719-741 AD-1230756 AAACUGUACCACAGUGAUGCA 252 804-824 UGCAUCACUGUGGUACAGUUUUU 552 802-824 AD-1230757 CACAGUGAUGCCUGUUCUUCA 253 813-833 UGAAGAACAGGCAUCACUGUGGU 553 811-833 AD-1230758 AACAAUGACAUUGCGCUGAUA 254 1161-1181 UAUCAGCGCAAUGUCAUUGUUCU 554 1159-1181 AD-1230759 CAAUGACAUUGCGCUGAUGAA 255 1163-1183 UUCAUCAGCGCAAUGUCAUUGUU 555 1161-1183 AD-1230760 AAAGAUGUGUUUUGUUUUGGA 256 2728-2748 UCCAAAACAAAACACAUCUUUCU 556 2726-2748 AD-1230761 UGCCAAGUGCCAUAACCAUGA 257 2809-2829 UCAUGGUUAUGGCACUUGGCAAU 557 2807-2829 AD-1230762 UGUGCACCUCAAAGACUAAGA 258 361-381 UCUUAGUCUUUGAGGUGCACACU 558 359-381 AD-1230763 AGACUAAGAAAGCACUGUGCA 259 373-393 UGCACAGUGCUUUCUUAGUCUUU 559 371-393 AD-1230764 AGGUGUACUCAUCUCAGAGGA 260 610-630 UCCUCUGAGAUGAGUACACCUGA 560 608-630 AD-1230765 CCACAGUGAUGCCUGUUCUUA 261 812-832 UAAGAACAGGCAUCACUGUGGUA 561 810-832 AD-1230766 ACAGUGAUGCCUGUUCUUCAA 262 814-834 UUGAAGAACAGGCAUCACUGUGG 562 812-834 AD-1230767 UGUUCUUCAAAAGCAGUGGUA 263 825-845 UACCACUGCUUUUGAAGAACAGG 563 823-845 AD-1230768 AGAACAAUGACAUUGCGCUGA 264 1159-1179 UCAGCGCAAUGUCAUUGUUCUUG 564 1157-1179 AD-1230769 AAGAUGGCUUUGAACUCAGGA 265 132-152 UCCUGAGUUCAAAGCCAUCUUGC 565 130-152 AD-1230770 GAUGGCUUUGAACUCAGGGUA 266 134-154 UACCCUGAGUUCAAAGCCAUCUU 566 132-154 AD-1230771 GCACUCUCUGCCAUUCUGUGA 267 1751-1771 UCACAGAAUGGCAGAGAGUGCCA 567 1749-1771 AD-1230772 GGUGACGUGGUAGUCACUUGA 268 2158-2178 UCAAGUGACUACCACGUCACCAC 568 2156-2178 AD-1230773 GUGACGUGGUAGUCACUUGUA 269 2159-2179 UACAAGUGACUACCACGUCACCA 569 2157-2179 AD-1230774 CUAGUCACUGGAAAUUGAGGA 270 2441-2461 UCCUCAAUUUCCAGUGACUAGCA 570 2439-2461 AD-1230775 ACUGGAAAUUGAGGUCCAUGA 271 2447-2467 UCAUGGACCUCAAUUUCCAGUGA 571 2445-2467 AD-1230776 GAAGAGAAAGAUGUGUUUUGA 272 2722-2742 UCAAAACACAUCUUUCUCUUCUU 572 2720-2742 AD-1230777 GAGAAAGAUGUGUUUUGUUUA 273 2725-2745 UAAACAAAACACAUCUUUCUCUU 573 2723-2745 AD-1230778 AAGAUGUGUUUUGUUUUGGAA 274 2729-2749 UUCCAAAACAAAACACAUCUUUC 574 2727-2749 AD-1230779 UUGUUUUGGACUCUCUGUGGA 275 2739-2759 UCCACAGAGAGUCCAAAACAAAA 575 2737-2759 AD-1230780 UGUACUCAUCUCAGAGGAAGA 276 613-633 UCUUCCUCUGAGAUGAGUACACC 576 611-633 AD-1230781 CCAAGAACAAUGACAUUGCGA 277 1156-1176 UCGCAAUGUCAUUGUUCUUGGUC 577 1154-1176 AD-1230782 GAACAAUGACAUUGCGCUGAA 278 1160-1180 UUCAGCGCAAUGUCAUUGUUCUU 578 1158-1180 AD-1230783 UGACAUUGCGCUGAUGAAGCA 279 1166-1186 UGCUUCAUCAGCGCAAUGUCAUU 579 1164-1186 AD-1230784 GGAGAAAGGGAAGACCUCAGA 280 1298-1318 UCUGAGGUCUUCCCUUUCUCCUC 580 1296-1318 AD-1230785 UUGGCACUCUCUGCCAUUCUA 281 1748-1768 UAGAAUGGCAGAGAGUGCCAAAG 581 1746-1768 AD-1230786 CGAAGAAGAGAAAGAUGUGUA 282 2718-2738 UACACAUCUUUCUCUUCUUCGCC 582 2716-2738 AD-1230787 AAGAGAAAGAUGUGUUUUGUA 283 2723-2743 UACAAAACACAUCUUUCUCUUCU 583 2721-2743 AD-1230788 AGGCUGGUUUGCAAGAAUGAA 284 2866-2886 UUCAUUCUUGCAAACCAGCCUGC 584 2864-2886 AD-1230789 UGCAAUCCCAUUUGCAGGAUA 285 2932-2952 UAUCCUGCAAAUGGGAUUGCAUG 585 2930-2952 AD-1230790 GCAAUCCCAUUUGCAGGAUCA 286 2933-2953 UGAUCCUGCAAAUGGGAUUGCAU 586 2931-2953 AD-1230791 CAUGCCUCUGUAGAGAGCAGA 287 2963-2983 UCUGCUCUCUACAGAGGCAUGUG 587 2961-2983 AD-1230792 UCUGGGAUAGAGUGCGACUCA 288 480-500 UGAGUCGCACUCUAUCCCAGAGU 588 478-500 AD-1230793 ACUCAUCUCAGAGGAAGUCCA 289 616-636 UGGACUUCCUCUGAGAUGAGUAC 589 614-636 AD-1230794 ACGACUGGAACGAGAACUACA 290 655-675 UGUAGUUCUCGUUCCAGUCGUCU 590 653-675 AD-1230795 UUUUUACUCUAGCCAAGGAAA 291 713-733 UUUCCUUGGCUAGAGUAAAAAUU 591 711-733 AD-1230796 UUACUCUAGCCAAGGAAUAGA 292 716-736 UCUAUUCCUUGGCUAGAGUAAAA 592 714-736 AD-1230797 ACUGUACCACAGUGAUGCCUA 293 806-826 UAGGCAUCACUGUGGUACAGUUU 593 804-826 AD-1230798 ACCACAGUGAUGCCUGUUCUA 294 811-831 UAGAACAGGCAUCACUGUGGUAC 594 809-831 AD-1230799 ACAAUGACAUUGCGCUGAUGA 295 1162-1182 UCAUCAGCGCAAUGUCAUUGUUC 595 1160-1182 AD-1230800 AUGACAUUGCGCUGAUGAAGA 296 1165-1185 UCUUCAUCAGCGCAAUGUCAUUG 596 1163-1185 AD-1230801 CCAGGAGUGUACGGGAAUGUA 297 1545-1565 UACAUUCCCGUACACUCCUGGUC 597 1543-1565 AD-1230802 UAUUCACGGACUGGAUUUAUA 298 1570-1590 UAUAAAUCCAGUCCGUGAAUACC 598 1568-1590 AD-1230803 GAUUUAUCGACAAAUGAGGGA 299 1583-1603 UCCCUCAUUUGUCGAUAAAUCCA 599 1581-1603 AD-1230804 AGAGAUGAUUCAGAGGUCACA 300 1695-1715 UGUGACCUCUGAAUCAUCUCUAA 600 1693-1715 AD-1230805 AGAGAAAGAUGUGUUUUGUUA 301 2724-2744 UAACAAAACACAUCUUUCUCUUC 601 2722-2744 AD-1230806 CAAGUGCCAUAACCAUGAGCA 302 2812-2832 UGCUCAUGGUUAUGGCACUUGGC 602 2810-2832 AD-1230807 CAUAACCAUGAGCACUACUCA 303 2819-2839 UGAGUAGUGCUCAUGGUUAUGGC 603 2817-2839 AD-1230808 CAUUUGCAGGAUCUGUCUGUA 304 2940-2960 UACAGACAGAUCCUGCAAAUGGG 604 2938-2960 AD-1230809 UCAUCUCAGAGGAAGUCCUGA 305 618-638 UCAGGACUUCCUCUGAGAUGAGU 605 616-638 AD-1230810 CAGGAGUGUACGGGAAUGUGA 306 1546-1566 UCACAUUCCCGUACACUCCUGGU 606 1544-1566 AD-1230811 UCUGGCUUUGGCACUCUCUGA 307 1741-1761 UCAGAGAGUGCCAAAGCCAGACA 607 1739-1761 AD-1230812 UGGCACUCUCUGCCAUUCUGA 308 1749-1769 UCAGAAUGGCAGAGAGUGCCAAA 608 1747-1769 AD-1230813 GGCACUCUCUGCCAUUCUGUA 309 1750-1770 UACAGAAUGGCAGAGAGUGCCAA 609 1748-1770 AD-1230814 ACCAUGGAUACCAACCGGAAA 310 184-204 UUUCCGGUUGGUAUCCAUGGUUU 610 182-204 AD-1230815 CCAUGGAUACCAACCGGAAAA 311 185-205 UUUUCCGGUUGGUAUCCAUGGUU 611 183-205 AD-1230816 GGGAAAUCAAGGAUGCUCAGA 312 2468-2488 UCUGAGCAUCCUUGAUUUCCCCC 612 2466-2488 AD-1230817 CCAAGUGCCAUAACCAUGAGA 313 2811-2831 UCUCAUGGUUAUGGCACUUGGCA 613 2809-2831 AD-1230818 CAUGCAAUCCCAUUUGCAGGA 314 2930-2950 UCCUGCAAAUGGGAUUGCAUGAC 614 2928-2950 AD-1230819 CCAUUUGCAGGAUCUGUCUGA 315 2939-2959 UCAGACAGAUCCUGCAAAUGGGA 615 2937-2959 AD-1230820 UGCAGGAUCUGUCUGUGCACA 316 2944-2964 UGUGCACAGACAGAUCCUGCAAA 616 2942-2964

TABLE 3 Modified Sense and Antisense Strand TMPRSS2 dsRNA Sequences SEQ SEQ ID ID Duplex Name Sense Sequence 5′ to 3′ NO: Antisense Sequence 5′ to 3′ NO: AD-1230521 ususcau(Uhd)UfaAfCfUfcuuugaaascsa 617 VPusGfsuuuCfaaagaguUfaAfaugaasgsg 917 AD-1230522 asasacg(Uhd)CfuUfCfCfuucuuuaususa 618 VPusAfsauaAfagaaggaAfgAfcguuususc 918 AD-1230523 asascgu(Chd)UfuCfCfUfucuuuauusgsa 619 VPusCfsaauAfaagaaggAfaGfacguususu 919 AD-1230524 usgsgugaAfaAfCfGfucuu(Chd)cuuscsa 620 VPusGfsaagGfaagacguUfuUfcaccasusu 920 AD-1230525 usgsguu(Uhd)CfuUfUfAfcgcuguausasa 621 VPusUfsauaCfagcguaaAfgAfaaccascsu 921 AD-1230526 ususccagAfuAfCfCfuau(Chd)auuascsa 622 VPusGfsuaaUfgauagguAfuCfuggaasusg 922 AD-1230527 uscscaga(Uhd)aCfCfUfaucauuacsusa 623 VPusAfsguaAfugauaggUfaUfcuggasasu 923 AD-1230528 csasgaua(Chd)cUfAfUfcauuacucsgsa 624 VPusCfsgagUfaaugauaGfgUfaucugsgsa 924 AD-1230529 gsasuac(Chd)UfaUfCfAfuuacucgasusa 625 VPusAfsucgAfguaaugaUfaGfguaucsusg 925 AD-1230530 csgsuggaAfaAfAfCfcucu(Uhd)aacsasa 626 VPusUfsguuAfagagguuUfuUfccacgscsa 926 AD-1230531 asgsuga(Uhd)UfuCfUfCfauccaaaususa 627 VPusAfsauuUfggaugagAfaAfucacususu 927 AD-1230532 uscsuca(Uhd)CfcAfAfAfuuaugacuscsa 628 VPusGfsaguCfauaauuuGfgAfugagasasa 928 AD-1230533 asusccaaAfuUfAfUfgacu(Chd)caasgsa 629 VPusCfsuugGfagucauaAfuUfuggausgsa 929 AD-1230534 ascsugga(Uhd)uUfAfUfcgacaaausgsa 630 VPusCfsauuUfgucgauaAfaUfccaguscsc 930 AD-1230535 ususuga(Uhd)GfuCfUfCfcaaguaguscsa 631 VPusGfsacuAfcuuggagAfcAfucaaasasg 931 AD-1230536 csasaga(Chd)AfcAfUfCfcuaaaaggsusa 632 VPusAfsccuUfuuaggauGfuGfucuugsgsg 932 AD-1230537 gsusaa(Uhd)gGfuGfAfAfaacgucuuscsa 633 VPusGfsaagAfcguuuucAfcCfauuacsasa 933 AD-1230538 usgsaaaa(Chd)gUfCfUfuccuucuususa 634 VPusAfsaagAfaggaagaCfgUfuuucascsc 934 AD-1230539 gsasaaa(Chd)GfuCfUfUfccuucuuusasa 635 VPusUfsaaaGfaaggaagAfcGfuuuucsasc 935 AD-1230540 asascug(Uhd)AfaAfGfUfucaauugusgsa 636 VPusCfsacaAfuugaacuUfuAfcaguususa 936 AD-1230541 gsusacu(Chd)AfuCfUfCfagaggaagsusa 637 VPusAfscuuCfcucugagAfuGfaguacsasc 937 AD-1230542 csgsgcaa(Uhd)gUfCfGfauaucuausasa 638 VPusUfsauaGfauaucgaCfaUfugccgsgsc 938 AD-1230543 gsgscaa(Uhd)GfuCfGfAfuaucuauasasa 639 VPusUfsuauAfgauaucgAfcAfuugccsgsg 939 AD-1230544 gscsaaug(Uhd)cGfAfUfaucuauaasasa 640 VPusUfsuuaUfagauaucGfaCfauugcscsg 940 AD-1230545 csasaug(Uhd)CfgAfUfAfucuauaaasasa 641 VPusUfsuuuAfuagauauCfgAfcauugscsc 941 AD-1230546 gsusggu(Uhd)UfcUfUfUfacgcuguasusa 642 VPusAfsuacAfgcguaaaGfaAfaccacsusg 942 AD-1230547 asusuc(Chd)aGfaUfAfCfcuaucauusasa 643 VPusUfsaauGfauagguaUfcUfggaausgsu 943 AD-1230548 cscsaga(Uhd)AfcCfUfAfucauuacuscsa 644 VPusGfsaguAfaugauagGfuAfucuggsasa 944 AD-1230549 asgsaua(Chd)CfuAfUfCfauuacucgsasa 645 VPusUfscgaGfuaaugauAfgGfuaucusgsg 945 AD-1230550 gsusgau(Uhd)UfcUfCfAfuccaaauusasa 646 VPusUfsaauUfuggaugaGfaAfaucacsusu 946 AD-1230551 usgsauu(Uhd)CfuCfAfUfccaaauuasusa 647 VPusAfsuaaUfuuggaugAfgAfaaucascsu 947 AD-1230552 asusuuc(Uhd)CfaUfCfCfaaauuaugsasa 648 VPusUfscauAfauuuggaUfgAfgaaauscsa 948 AD-1230553 ususucu(Chd)AfuCfCfAfaauuaugascsa 649 VPusGfsucaUfaauuuggAfuGfagaaasusc 949 AD-1230554 ususcuca(Uhd)cCfAfAfauuaugacsusa 650 VPusAfsgucAfuaauuugGfaUfgagaasasu 950 AD-1230555 asgsguca(Chd)uUfCfAfuuuuuauusasa 651 VPusUfsaauAfaaaaugaAfgUfgaccuscsu 951 AD-1230556 asusguu(Uhd)CfuAfCfAfcauugcuascsa 652 VPusGfsuagCfaauguguAfgAfaacausasa 952 AD-1230557 uscsauu(Uhd)AfaCfUfCfuuugaaacsusa 653 VPusAfsguuUfcaaagagUfuAfaaugasasg 953 AD-1230558 csusagga(Chd)uUfAfAfccuugaaasusa 654 VPusAfsuuuCfaagguuaAfgUfccuagscsu 954 AD-1230559 asgsaca(Chd)AfuCfCfUfaaaaggugsusa 655 VPusAfscacCfuuuuaggAfuGfugucususg 955 AD-1230560 gsascaca(Uhd)cCfUfAfaaaggugususa 656 VPusAfsacaCfcuuuuagGfaUfgugucsusu 956 AD-1230561 asusgg(Uhd)gAfaAfAfCfgucuuccususa 657 VPusAfsaggAfagacguuUfuCfaccaususa 957 AD-1230562 gsusgaaaAfcGfUfCfuucc(Uhd)ucususa 658 VPusAfsagaAfggaagacGfuUfuucacscsa 958 AD-1230563 asasaacg(Uhd)cUfUfCfcuucuuuasusa 659 VPusAfsuaaAfgaaggaaGfaCfguuuuscsa 959 AD-1230564 asusgcc(Uhd)GfuUfCfUfucaaaagcsasa 660 VPusUfsgcuUfuugaagaAfcAfggcauscsa 960 AD-1230565 gscscug(Uhd)UfcUfUfCfaaaagcagsusa 661 VPusAfscugCfuuuugaaGfaAfcaggcsasu 961 AD-1230566 ususcaaaAfgCfAfGfuggu(Uhd)ucususa 662 VPusAfsagaAfaccacugCfuUfuugaasgsa 962 AD-1230567 csusauca(Uhd)uAfCfUfcgaugcugsusa 663 VPusAfscagCfaucgaguAfaUfgauagsgsu 963 AD-1230568 usgscg(Uhd)gGfaAfAfAfaccucuuasasa 664 VPusUfsuaaGfagguuuuUfcCfacgcasgsu 964 AD-1230569 csasag(Uhd)aGfaAfAfAfagugauuuscsa 665 VPusGfsaaaUfcacuuuuUfcUfacuugsgsu 965 AD-1230570 asasguagAfaAfAfAfguga(Uhd)uucsusa 666 VPusAfsgaaAfucacuuuUfuCfuacuusgsg 966 AD-1230571 gsusagaaAfaAfGfUfgauu(Uhd)cucsasa 667 VPusUfsgagAfaaucacuUfuUfucuacsusu 967 AD-1230572 usasgaaaAfaGfUfGfauuu(Chd)ucasusa 668 VPusAfsugaGfaaaucacUfuUfuucuascsu 968 AD-1230573 asasaag(Uhd)GfaUfUfUfcucauccasasa 669 VPusUfsuggAfugagaaaUfcAfcuuuususc 969 AD-1230574 gsasuuu(Chd)UfcAfUfCfcaaauuausgsa 670 VPusCfsauaAfuuuggauGfaGfaaaucsasc 970 AD-1230575 asasgcc(Uhd)CfuGfAfCfuuucaacgsasa 671 VPusUfscguUfgaaagucAfgAfggcuuscsu 971 AD-1230576 gscsuag(Uhd)CfaCfUfGfgaaauugasgsa 672 VPusCfsucaAfuuuccagUfgAfcuagcsasg 972 AD-1230577 ususuuga(Uhd)gUfCfUfccaaguagsusa 673 VPusAfscuaCfuuggagaCfaUfcaaaasgsc 973 AD-1230578 gsasugug(Uhd)uUfUfGfuuuuggacsusa 674 VPusAfsgucCfaaaacaaAfaCfacaucsusu 974 AD-1230579 ususuug(Uhd)UfuUfGfGfacucucugsusa 675 VPusAfscagAfgaguccaAfaAfcaaaascsa 975 AD-1230580 gscsugg(Uhd)UfuGfCfAfagaaugaasasa 676 VPusUfsuucAfuucuugcAfaAfccagcscsu 976 AD-1230581 usgsguu(Uhd)GfcAfAfGfaaugaaausgsa 677 VPusCfsauuUfcauucuuGfcAfaaccasgsc 977 AD-1230582 gsgsuuug(Chd)aAfGfAfaugaaaugsasa 678 VPusUfscauUfucauucuUfgCfaaaccsasg 978 AD-1230583 usasgga(Chd)UfuAfAfCfcuugaaausgsa 679 VPusCfsauuUfcaagguuAfaGfuccuasgsc 979 AD-1230584 gsgsugaaAfaCfGfUfcuuc(Chd)uucsusa 680 VPusAfsgaaGfgaagacgUfuUfucaccsasu 980 AD-1230585 ascsguc(Uhd)UfcCfUfUfcuuuauugscsa 681 VPusGfscaaUfaaagaagGfaAfgacgususu 981 AD-1230586 csgsucu(Uhd)CfcUfUfCfuuuauugcscsa 682 VPusGfsgcaAfuaaagaaGfgAfagacgsusu 982 AD-1230587 csusguaaAfgUfUfCfaau(Uhd)gugasasa 683 VPusUfsucaCfaauugaaCfuUfuacagsusu 983 AD-1230588 gscsacc(Uhd)CfaAfAfGfacuaagaasasa 684 VPusUfsuucUfuagucuuUfgAfggugcsasc 984 AD-1230589 csasccu(Chd)AfaAfGfAfcuaagaaasgsa 685 VPusCfsuuuCfuuagucuUfuGfaggugscsa 985 AD-1230590 asuscca(Chd)CfaGfCfUfuuaugaaascsa 686 VPusGfsuuuCfauaaagcUfgGfuggauscsc 986 AD-1230591 usgsaug(Chd)CfuGfUfUfcuucaaaasgsa 687 VPusCfsuuuUfgaagaacAfgGfcaucascsu 987 AD-1230592 gsasugc(Chd)UfgUfUfCfuucaaaagscsa 688 VPusGfscuuUfugaagaaCfaGfgcaucsasc 988 AD-1230593 usgsccug(Uhd)uCfUfUfcaaaagcasgsa 689 VPusCfsugcUfuuugaagAfaCfaggcasusc 989 AD-1230594 uscsuu(Chd)aAfaAfGfCfagugguuuscsa 690 VPusGfsaaaCfcacugcuUfuUfgaagasasc 990 AD-1230595 csasuuc(Chd)AfgAfUfAfccuaucaususa 691 VPusAfsaugAfuagguauCfuGfgaaugsusu 991 AD-1230596 ascscua(Uhd)CfaUfUfAfcucgaugcsusa 692 VPusAfsgcaUfcgaguaaUfgAfuaggusasu 992 AD-1230597 gscsguggAfaAfAfAfccuc(Uhd)uaascsa 693 VPusGfsuuaAfgagguuuUfuCfcacgcsasg 993 AD-1230598 csasuua(Chd)UfcGfAfUfgcuguugasusa 694 VPusAfsucaAfcagcaucGfaGfuaaugsasu 994 AD-1230599 ususacu(Chd)GfaUfGfCfuguugauasasa 695 VPusUfsuauCfaacagcaUfcGfaguaasusg 995 AD-1230600 asasgaa(Chd)AfaUfGfAfcauugcgcsusa 696 VPusAfsgcgCfaaugucaUfuGfuucuusgsg 996 AD-1230601 ascsag(Chd)aAfgAfUfGfgcuuugaascsa 697 VPusGfsuucAfaagccauCfuUfgcugususa 997 AD-1230602 csasgcaaGfaUfGfGfcuu(Uhd)gaacsusa 698 VPusAfsguuCfaaagccaUfcUfugcugsusu 998 AD-1230603 uscsagagGfuCfAfCfuuca(Uhd)uuususa 699 VPusAfsaaaAfugaagugAfcCfucugasasu 999 AD-1230604 csasgagg(Uhd)cAfCfUfucauuuuusasa 700 VPusUfsaaaAfaugaaguGfaCfcucugsasa 1000 AD-1230605 usgsuua(Uhd)GfuUfUfCfuacacauusgsa 701 VPusCfsaauGfuguagaaAfcAfuaacasusg 1001 AD-1230606 usasugu(Uhd)UfcUfAfCfacauugcusasa 702 VPusUfsagcAfauguguaGfaAfacauasasc 1002 AD-1230607 ascsguu(Chd)UfaUfAfAfaugaaugusgsa 703 VPusCfsacaUfucauuuaUfaGfaacgususa 1003 AD-1230608 gsusuuug(Uhd)uUfUfGfgacucucusgsa 704 VPusCfsagaGfaguccaaAfaCfaaaacsasc 1004 AD-1230609 gsusuug(Chd)AfaGfAfAfugaaaugasasa 705 VPusUfsucaUfuucauucUfuGfcaaacscsa 1005 AD-1230610 asasgaa(Uhd)GfaAfAfUfgaaugauuscsa 706 VPusGfsaauCfauucauuUfcAfuucuusgsc 1006 AD-1230611 gscsuaggAfcUfUfAfaccu(Uhd)gaasasa 707 VPusUfsuucAfagguuaaGfuCfcuagcsusg 1007 AD-1230612 asasgaca(Chd)aUfCfCfuaaaaggusgsa 708 VPusCfsaccUfuuuaggaUfgUfgucuusgsg 1008 AD-1230613 usgsuaa(Uhd)GfgUfGfAfaaacgucususa 709 VPusAfsagaCfguuuucaCfcAfuuacasasc 1009 AD-1230614 asasugg(Uhd)GfaAfAfAfcgucuuccsusa 710 VPusAfsggaAfgacguuuUfcAfccauusasc 1010 AD-1230615 ascsug(Uhd)aAfaGfUfUfcaauugugsasa 711 VPusUfscacAfauugaacUfuUfacagususu 1011 AD-1230616 usgsuaaaGfuUfCfAfauug(Uhd)gaasasa 712 VPusUfsuucAfcaauugaAfcUfuuacasgsu 1012 AD-1230617 ascscu(Chd)aAfaGfAfCfuaagaaagscsa 713 VPusGfscuuUfcuuagucUfuUfgaggusgsc 1013 AD-1230618 cscsu(Chd)aaAfgAfCfUfaagaaagcsasa 714 VPusUfsgcuUfucuuaguCfuUfugaggsusg 1014 AD-1230619 csus(Chd)aaaGfaCfUfAfagaaagcascsa 715 VPusGfsugcUfuucuuagUfcUfuugagsgsu 1015 AD-1230620 csasccag(Chd)uUfUfAfugaaacugsasa 716 VPusUfscagUfuucauaaAfgCfuggugsgsa 1016 AD-1230621 cscsgg(Chd)aAfuGfUfCfgauaucuasusa 717 VPusAfsuagAfuaucgacAfuUfgccggscsa 1017 AD-1230622 gsusucu(Uhd)CfaAfAfAfgcaguggususa 718 VPusAfsaccAfcugcuuuUfgAfagaacsasg 1018 AD-1230623 csusucaaAfaGfCfAfgugg(Uhd)uucsusa 719 VPusAfsgaaAfccacugcUfuUfugaagsasa 1019 AD-1230624 ususgaa(Chd)AfuUfCfCfagauaccusasa 720 VPusUfsaggUfaucuggaAfuGfuucaasusa 1020 AD-1230625 ascsauu(Chd)CfaGfAfUfaccuaucasusa 721 VPusAfsugaUfagguaucUfgGfaaugususc 1021 AD-1230626 uscsauua(Chd)uCfGfAfugcuguugsasa 722 VPusUfscaaCfagcaucgAfgUfaaugasusa 1022 AD-1230627 asgsuagaAfaAfAfGfugau(Uhd)ucuscsa 723 VPusGfsagaAfaucacuuUfuUfcuacususg 1023 AD-1230628 csusccaaGfaCfCfAfagaa(Chd)aausgsa 724 VPusCfsauuGfuucuuggUfcUfuggagsusc 1024 AD-1230629 csasaga(Chd)CfaAfGfAfacaaugacsasa 725 VPusUfsgucAfuuguucuUfgGfucuugsgsa 1025 AD-1230630 csasaga(Uhd)GfgCfUfUfugaacucasgsa 726 VPusCfsugaGfuucaaagCfcAfucuugscsu 1026 AD-1230631 gsascg(Uhd)gGfuAfGfUfcacuuguasasa 727 VPusUfsuacAfagugacuAfcCfacgucsasc 1027 AD-1230632 gsusuaug(Uhd)uUfCfUfacacauugscsa 728 VPusGfscaaUfguguagaAfaCfauaacsasu 1028 AD-1230633 ususug(Chd)aAfgAfAfUfgaaaugaasusa 729 VPusAfsuucAfuuucauuCfuUfgcaaascsc 1029 AD-1230634 asgsgac(Uhd)UfaAfCfCfuugaaaugsgsa 730 VPusCfscauUfucaagguUfaAfguccusasg 1030 AD-1230635 usgsugaaAfaUfGfAfauau(Chd)augscsa 731 VPusGfscauGfauauucaUfuUfucacasasu 1031 AD-1230636 asgsuga(Uhd)GfcCfUfGfuucuucaasasa 732 VPusUfsuugAfagaacagGfcAfucacusgsu 1032 AD-1230637 ususcuu(Chd)AfaAfAfGfcagugguususa 733 VPusAfsaacCfacugcuuUfuGfaagaascsa 1033 AD-1230638 uscsaaaaGfcAfGfUfgguu(Uhd)cuususa 734 VPusAfsaagAfaaccacuGfcUfuuugasasg 1034 AD-1230639 csasaaag(Chd)aGfUfGfguuucuuusasa 735 VPusUfsaaaGfaaaccacUfgCfuuuugsasa 1035 AD-1230640 gsgsuuu(Chd)UfuUfAfCfgcuguauasgsa 736 VPusCfsuauAfcagcguaAfaGfaaaccsasc 1036 AD-1230641 asusacc(Uhd)AfuCfAfUfuacucgausgsa 737 VPusCfsaucGfaguaaugAfuAfgguauscsu 1037 AD-1230642 asuscau(Uhd)AfcUfCfGfaugcuguusgsa 738 VPusCfsaacAfgcaucgaGfuAfaugausasg 1038 AD-1230643 asasaaag(Uhd)gAfUfUfucucauccsasa 739 VPusUfsggaUfgagaaauCfaCfuuuuuscsu 1039 AD-1230644 ascsuc(Chd)aAfgAfCfCfaagaacaasusa 740 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864 VPusCfsagcGfcaaugucAfuUfguucususg 1164 AD-1230769 asasga(Uhd)gGfcUfUfUfgaacucagsgsa 865 VPusCfscugAfguucaaaGfcCfaucuusgsc 1165 AD-1230770 gsasugg(Chd)UfuUfGfAfacucagggsusa 866 VPusAfscccUfgaguucaAfaGfccaucsusu 1166 AD-1230771 gscsacu(Chd)UfcUfGfCfcauucugusgsa 867 VPusCfsacaGfaauggcaGfaGfagugcscsa 1167 AD-1230772 gsgsuga(Chd)GfuGfGfUfagucacuusgsa 868 VPusCfsaagUfgacuaccAfcGfucaccsasc 1168 AD-1230773 gsusgacg(Uhd)gGfUfAfgucacuugsusa 869 VPusAfscaaGfugacuacCfaCfgucacscsa 1169 AD-1230774 csusagu(Chd)AfcUfGfGfaaauugagsgsa 870 VPusCfscucAfauuuccaGfuGfacuagscsa 1170 AD-1230775 ascsuggaAfaUfUfGfaggu(Chd)causgsa 871 VPusCfsaugGfaccucaaUfuUfccagusgsa 1171 AD-1230776 gsasagagAfaAfGfAfugug(Uhd)uuusgsa 872 VPusCfsaaaAfcacaucuUfuCfucuucsusu 1172 AD-1230777 gsasgaaaGfaUfGfUfguuu(Uhd)guususa 873 VPusAfsaacAfaaacacaUfcUfuucucsusu 1173 AD-1230778 asasgaug(Uhd)gUfUfUfuguuuuggsasa 874 VPusUfsccaAfaacaaaaCfaCfaucuususc 1174 AD-1230779 ususguu(Uhd)UfgGfAfCfucucugugsgsa 875 VPusCfscacAfgagagucCfaAfaacaasasa 1175 AD-1230780 usgsuac(Uhd)CfaUfCfUfcagaggaasgsa 876 VPusCfsuucCfucugagaUfgAfguacascsc 1176 AD-1230781 cscsaagaAfcAfAfUfgaca(Uhd)ugcsgsa 877 VPusCfsgcaAfugucauuGfuUfcuuggsusc 1177 AD-1230782 gsasacaa(Uhd)gAfCfAfuugcgcugsasa 878 VPusUfscagCfgcaauguCfaUfuguucsusu 1178 AD-1230783 usgsaca(Uhd)UfgCfGfCfugaugaagscsa 879 VPusGfscuuCfaucagcgCfaAfugucasusu 1179 AD-1230784 gsgsagaaAfgGfGfAfagac(Chd)ucasgsa 880 VPusCfsugaGfgucuuccCfuUfucuccsusc 1180 AD-1230785 ususggca(Chd)uCfUfCfugccauucsusa 881 VPusAfsgaaUfggcagagAfgUfgccaasasg 1181 AD-1230786 csgsaagaAfgAfGfAfaaga(Uhd)gugsusa 882 VPusAfscacAfucuuucuCfuUfcuucgscsc 1182 AD-1230787 asasgagaAfaGfAfUfgugu(Uhd)uugsusa 883 VPusAfscaaAfacacaucUfuUfcucuuscsu 1183 AD-1230788 asgsgc(Uhd)gGfuUfUfGfcaagaaugsasa 884 VPusUfscauUfcuugcaaAfcCfagccusgsc 1184 AD-1230789 usgscaa(Uhd)CfcCfAfUfuugcaggasusa 885 VPusAfsuccUfgcaaaugGfgAfuugcasusg 1185 AD-1230790 gscsaau(Chd)CfcAfUfUfugcaggauscsa 886 VPusGfsaucCfugcaaauGfgGfauugcsasu 1186 AD-1230791 csasugc(Chd)UfcUfGfUfagagagcasgsa 887 VPusCfsugcUfcucuacaGfaGfgcaugsusg 1187 AD-1230792 uscsugggAfuAfGfAfgug(Chd)gacuscsa 888 VPusGfsaguCfgcacucuAfuCfccagasgsu 1188 AD-1230793 ascsuca(Uhd)CfuCfAfGfaggaagucscsa 889 VPusGfsgacUfuccucugAfgAfugagusasc 1189 AD-1230794 ascsgac(Uhd)GfgAfAfCfgagaacuascsa 890 VPusGfsuagUfucucguuCfcAfgucguscsu 1190 AD-1230795 ususuuua(Chd)uCfUfAfgccaaggasasa 891 VPusUfsuccUfuggcuagAfgUfaaaaasusu 1191 AD-1230796 ususacu(Chd)UfaGfCfCfaaggaauasgsa 892 VPusCfsuauUfccuuggcUfaGfaguaasasa 1192 AD-1230797 ascsugua(Chd)cAfCfAfgugaugccsusa 893 VPusAfsggcAfucacuguGfgUfacagususu 1193 AD-1230798 ascsca(Chd)aGfuGfAfUfgccuguucsusa 894 VPusAfsgaaCfaggcaucAfcUfguggusasc 1194 AD-1230799 ascsaa(Uhd)gAfcAfUfUfgcgcugausgsa 895 VPusCfsaucAfgcgcaauGfuCfauugususc 1195 AD-1230800 asusgaca(Uhd)uGfCfGfcugaugaasgsa 896 VPusCfsuucAfucagcgcAfaUfgucaususg 1196 AD-1230801 cscsaggaGfuGfUfAfcgggaa(Uhd)gsusa 897 VPusAfscauUfcccguacAfcUfccuggsusc 1197 AD-1230802 usasuuca(Chd)gGfAfCfuggauuuasusa 898 VPusAfsuaaAfuccagucCfgUfgaauascsc 1198 AD-1230803 gsasuuua(Uhd)cGfAfCfaaaugaggsgsa 899 VPusCfsccuCfauuugucGfaUfaaaucscsa 1199 AD-1230804 asgsaga(Uhd)GfaUfUfCfagaggucascsa 900 VPusGfsugaCfcucugaaUfcAfucucusasa 1200 AD-1230805 asgsagaaAfgAfUfGfuguu(Uhd)ugususa 901 VPusAfsacaAfaacacauCfuUfucucususc 1201 AD-1230806 csasagug(Chd)cAfUfAfaccaugagscsa 902 VPusGfscucAfugguuauGfgCfacuugsgsc 1202 AD-1230807 csasuaa(Chd)CfaUfGfAfgcacuacuscsa 903 VPusGfsaguAfgugcucaUfgGfuuaugsgsc 1203 AD-1230808 csasuuug(Chd)aGfGfAfucugucugsusa 904 VPusAfscagAfcagauccUfgCfaaaugsgsg 1204 AD-1230809 uscsauc(Uhd)CfaGfAfGfgaaguccusgsa 905 VPusCfsaggAfcuuccucUfgAfgaugasgsu 1205 AD-1230810 csasggag(Uhd)gUfAfCfgggaaugusgsa 906 VPusCfsacaUfucccguaCfaCfuccugsgsu 1206 AD-1230811 uscsugg(Chd)UfuUfGfGfcacucucusgsa 907 VPusCfsagaGfagugccaAfaGfccagascsa 1207 AD-1230812 usgsgca(Chd)UfcUfCfUfgccauucusgsa 908 VPusCfsagaAfuggcagaGfaGfugccasasa 1208 AD-1230813 gsgscac(Uhd)CfuCfUfGfccauucugsusa 909 VPusAfscagAfauggcagAfgAfgugccsasa 1209 AD-1230814 ascsca(Uhd)gGfaUfAfCfcaaccggasasa 910 VPusUfsuccGfguugguaUfcCfauggususu 1210 AD-1230815 cscsauggAfuAfCfCfaac(Chd)ggaasasa 911 VPusUfsuucCfgguugguAfuCfcauggsusu 1211 AD-1230816 gsgsgaaa(Uhd)cAfAfGfgaugcucasgsa 912 VPusCfsugaGfcauccuuGfaUfuucccscsc 1212 AD-1230817 cscsaag(Uhd)GfcCfAfUfaaccaugasgsa 913 VPusCfsucaUfgguuaugGfcAfcuuggscsa 1213 AD-1230818 csasug(Chd)aAfuCfCfCfauuugcagsgsa 914 VPusCfscugCfaaaugggAfuUfgcaugsasc 1214 AD-1230819 cscsauu(Uhd)GfcAfGfGfaucugucusgsa 915 VPusCfsagaCfagauccuGfcAfaauggsgsa 1215 AD-1230820 usgscaggAfuCfUfGfucug(Uhd)gcascsa 916 VPusGfsugcAfcagacagAfuCfcugcasasa 1216 SEQ ID Duplex Name mRNA target sequence 5′ to 3′ NO: AD-1230521 CCUUCAUUUAACUCUUUGAAACU 1217 AD-1230522 GAAAACGUCUUCCUUCUUUAUUG 1218 AD-1230523 AAAACGUCUUCCUUCUUUAUUGC 1219 AD-1230524 AAUGGUGAAAACGUCUUCCUUCU 1220 AD-1230525 AGUGGUUUCUUUACGCUGUAUAG 1221 AD-1230526 CAUUCCAGAUACCUAUCAUUACU 1222 AD-1230527 AUUCCAGAUACCUAUCAUUACUC 1223 AD-1230528 UCCAGAUACCUAUCAUUACUCGA 1224 AD-1230529 CAGAUACCUAUCAUUACUCGAUG 1225 AD-1230530 UGCGUGGAAAAACCUCUUAACAA 1226 AD-1230531 AAAGUGAUUUCUCAUCCAAAUUA 1227 AD-1230532 UUUCUCAUCCAAAUUAUGACUCC 1228 AD-1230533 UCAUCCAAAUUAUGACUCCAAGA 1229 AD-1230534 GGACUGGAUUUAUCGACAAAUGA 1230 AD-1230535 CUUUUGAUGUCUCCAAGUAGUCC 1231 AD-1230536 CCCAAGACACAUCCUAAAAGGUG 1232 AD-1230537 UUGUAAUGGUGAAAACGUCUUCC 1233 AD-1230538 GGUGAAAACGUCUUCCUUCUUUA 1234 AD-1230539 GUGAAAACGUCUUCCUUCUUUAU 1235 AD-1230540 UAAACUGUAAAGUUCAAUUGUGA 1236 AD-1230541 GUGUACUCAUCUCAGAGGAAGUC 1237 AD-1230542 GCCGGCAAUGUCGAUAUCUAUAA 1238 AD-1230543 CCGGCAAUGUCGAUAUCUAUAAA 1239 AD-1230544 CGGCAAUGUCGAUAUCUAUAAAA 1240 AD-1230545 GGCAAUGUCGAUAUCUAUAAAAA 1241 AD-1230546 CAGUGGUUUCUUUACGCUGUAUA 1242 AD-1230547 ACAUUCCAGAUACCUAUCAUUAC 1243 AD-1230548 UUCCAGAUACCUAUCAUUACUCG 1244 AD-1230549 CCAGAUACCUAUCAUUACUCGAU 1245 AD-1230550 AAGUGAUUUCUCAUCCAAAUUAU 1246 AD-1230551 AGUGAUUUCUCAUCCAAAUUAUG 1247 AD-1230552 UGAUUUCUCAUCCAAAUUAUGAC 1248 AD-1230553 GAUUUCUCAUCCAAAUUAUGACU 1249 AD-1230554 AUUUCUCAUCCAAAUUAUGACUC 1250 AD-1230555 AGAGGUCACUUCAUUUUUAUUAA 1251 AD-1230556 UUAUGUUUCUACACAUUGCUACC 1252 AD-1230557 CUUCAUUUAACUCUUUGAAACUG 1253 AD-1230558 AGCUAGGACUUAACCUUGAAAUG 1254 AD-1230559 CAAGACACAUCCUAAAAGGUGUU 1255 AD-1230560 AAGACACAUCCUAAAAGGUGUUG 1256 AD-1230561 UAAUGGUGAAAACGUCUUCCUUC 1257 AD-1230562 UGGUGAAAACGUCUUCCUUCUUU 1258 AD-1230563 UGAAAACGUCUUCCUUCUUUAUU 1259 AD-1230564 UGAUGCCUGUUCUUCAAAAGCAG 1260 AD-1230565 AUGCCUGUUCUUCAAAAGCAGUG 1261 AD-1230566 UCUUCAAAAGCAGUGGUUUCUUU 1262 AD-1230567 ACCUAUCAUUACUCGAUGCUGUU 1263 AD-1230568 ACUGCGUGGAAAAACCUCUUAAC 1264 AD-1230569 ACCAAGUAGAAAAAGUGAUUUCU 1265 AD-1230570 CCAAGUAGAAAAAGUGAUUUCUC 1266 AD-1230571 AAGUAGAAAAAGUGAUUUCUCAU 1267 AD-1230572 AGUAGAAAAAGUGAUUUCUCAUC 1268 AD-1230573 GAAAAAGUGAUUUCUCAUCCAAA 1269 AD-1230574 GUGAUUUCUCAUCCAAAUUAUGA 1270 AD-1230575 AGAAGCCUCUGACUUUCAACGAC 1271 AD-1230576 CUGCUAGUCACUGGAAAUUGAGG 1272 AD-1230577 GCUUUUGAUGUCUCCAAGUAGUC 1273 AD-1230578 AAGAUGUGUUUUGUUUUGGACUC 1274 AD-1230579 UGUUUUGUUUUGGACUCUCUGUG 1275 AD-1230580 AGGCUGGUUUGCAAGAAUGAAAU 1276 AD-1230581 GCUGGUUUGCAAGAAUGAAAUGA 1277 AD-1230582 CUGGUUUGCAAGAAUGAAAUGAA 1278 AD-1230583 GCUAGGACUUAACCUUGAAAUGG 1279 AD-1230584 AUGGUGAAAACGUCUUCCUUCUU 1280 AD-1230585 AAACGUCUUCCUUCUUUAUUGCC 1281 AD-1230586 AACGUCUUCCUUCUUUAUUGCCC 1282 AD-1230587 AACUGUAAAGUUCAAUUGUGAAA 1283 AD-1230588 GUGCACCUCAAAGACUAAGAAAG 1284 AD-1230589 UGCACCUCAAAGACUAAGAAAGC 1285 AD-1230590 GGAUCCACCAGCUUUAUGAAACU 1286 AD-1230591 AGUGAUGCCUGUUCUUCAAAAGC 1287 AD-1230592 GUGAUGCCUGUUCUUCAAAAGCA 1288 AD-1230593 GAUGCCUGUUCUUCAAAAGCAGU 1289 AD-1230594 GUUCUUCAAAAGCAGUGGUUUCU 1290 AD-1230595 AACAUUCCAGAUACCUAUCAUUA 1291 AD-1230596 AUACCUAUCAUUACUCGAUGCUG 1292 AD-1230597 CUGCGUGGAAAAACCUCUUAACA 1293 AD-1230598 AUCAUUACUCGAUGCUGUUGAUA 1294 AD-1230599 CAUUACUCGAUGCUGUUGAUAAC 1295 AD-1230600 CCAAGAACAAUGACAUUGCGCUG 1296 AD-1230601 UAACAGCAAGAUGGCUUUGAACU 1297 AD-1230602 AACAGCAAGAUGGCUUUGAACUC 1298 AD-1230603 AUUCAGAGGUCACUUCAUUUUUA 1299 AD-1230604 UUCAGAGGUCACUUCAUUUUUAU 1300 AD-1230605 CAUGUUAUGUUUCUACACAUUGC 1301 AD-1230606 GUUAUGUUUCUACACAUUGCUAC 1302 AD-1230607 UAACGUUCUAUAAAUGAAUGUGC 1303 AD-1230608 GUGUUUUGUUUUGGACUCUCUGU 1304 AD-1230609 UGGUUUGCAAGAAUGAAAUGAAU 1305 AD-1230610 GCAAGAAUGAAAUGAAUGAUUCU 1306 AD-1230611 CAGCUAGGACUUAACCUUGAAAU 1307 AD-1230612 CCAAGACACAUCCUAAAAGGUGU 1308 AD-1230613 GUUGUAAUGGUGAAAACGUCUUC 1309 AD-1230614 GUAAUGGUGAAAACGUCUUCCUU 1310 AD-1230615 AAACUGUAAAGUUCAAUUGUGAA 1311 AD-1230616 ACUGUAAAGUUCAAUUGUGAAAA 1312 AD-1230617 GCACCUCAAAGACUAAGAAAGCA 1313 AD-1230618 CACCUCAAAGACUAAGAAAGCAC 1314 AD-1230619 ACCUCAAAGACUAAGAAAGCACU 1315 AD-1230620 UCCACCAGCUUUAUGAAACUGAA 1316 AD-1230621 UGCCGGCAAUGUCGAUAUCUAUA 1317 AD-1230622 CUGUUCUUCAAAAGCAGUGGUUU 1318 AD-1230623 UUCUUCAAAAGCAGUGGUUUCUU 1319 AD-1230624 UAUUGAACAUUCCAGAUACCUAU 1320 AD-1230625 GAACAUUCCAGAUACCUAUCAUU 1321 AD-1230626 UAUCAUUACUCGAUGCUGUUGAU 1322 AD-1230627 CAAGUAGAAAAAGUGAUUUCUCA 1323 AD-1230628 GACUCCAAGACCAAGAACAAUGA 1324 AD-1230629 UCCAAGACCAAGAACAAUGACAU 1325 AD-1230630 AGCAAGAUGGCUUUGAACUCAGG 1326 AD-1230631 GUGACGUGGUAGUCACUUGUAAG 1327 AD-1230632 AUGUUAUGUUUCUACACAUUGCU 1328 AD-1230633 GGUUUGCAAGAAUGAAAUGAAUG 1329 AD-1230634 CUAGGACUUAACCUUGAAAUGGA 1330 AD-1230635 AUUGUGAAAAUGAAUAUCAUGCA 1331 AD-1230636 ACAGUGAUGCCUGUUCUUCAAAA 1332 AD-1230637 UGUUCUUCAAAAGCAGUGGUUUC 1333 AD-1230638 CUUCAAAAGCAGUGGUUUCUUUA 1334 AD-1230639 UUCAAAAGCAGUGGUUUCUUUAC 1335 AD-1230640 GUGGUUUCUUUACGCUGUAUAGC 1336 AD-1230641 AGAUACCUAUCAUUACUCGAUGC 1337 AD-1230642 CUAUCAUUACUCGAUGCUGUUGA 1338 AD-1230643 AGAAAAAGUGAUUUCUCAUCCAA 1339 AD-1230644 UGACUCCAAGACCAAGAACAAUG 1340 AD-1230645 CUCCAAGACCAAGAACAAUGACA 1341 AD-1230646 CCAAGACCAAGAACAAUGACAUU 1342 AD-1230647 GACUGGAUUUAUCGACAAAUGAG 1343 AD-1230648 GAUUCAGAGGUCACUUCAUUUUU 1344 AD-1230649 UCAGAGGUCACUUCAUUUUUAUU 1345 AD-1230650 CAGAGGUCACUUCAUUUUUAUUA 1346 AD-1230651 GGGGAACAGAAACAUUUUUGUUC 1347 AD-1230652 CCUGCUAGUCACUGGAAAUUGAG 1348 AD-1230653 UGUUAUGUUUCUACACAUUGCUA 1349 AD-1230654 AGCUUUUGAUGUCUCCAAGUAGU 1350 AD-1230655 UUUGAUGUCUCCAAGUAGUCCAC 1351 AD-1230656 CGUUCUAUAAAUGAAUGUGCUGA 1352 AD-1230657 CAAGAAUGAAAUGAAUGAUUCUA 1353 AD-1230658 AGAAUGAAAUGAAUGAUUCUACA 1354 AD-1230659 UAGGACUUAACCUUGAAAUGGAA 1355 AD-1230660 AGGACUUAACCUUGAAAUGGAAA 1356 AD-1230661 AAAGUCAUGCAAUCCCAUUUGCA 1357 AD-1230662 AAGUCAUGCAAUCCCAUUUGCAG 1358 AD-1230663 GUGUGCACCUCAAAGACUAAGAA 1359 AD-1230664 GAUCCACCAGCUUUAUGAAACUG 1360 AD-1230665 CCAGCUUUAUGAAACUGAACACA 1361 AD-1230666 UUAUGAAACUGAACACAAGUGCC 1362 AD-1230667 AUAUUGAACAUUCCAGAUACCUA 1363 AD-1230668 UCAAAAGCAGUGGUUUCUUUACG 1364 AD-1230669 UGGUUUCUUUACGCUGUAUAGCC 1365 AD-1230670 UCAUUACUCGAUGCUGUUGAUAA 1366 AD-1230671 AAGACCAAGAACAAUGACAUUGC 1367 AD-1230672 AGACCAAGAACAAUGACAUUGCG 1368 AD-1230673 ACAGCAAGAUGGCUUUGAACUCA 1369 AD-1230674 UUCACGGACUGGAUUUAUCGACA 1370 AD-1230675 UCACGGACUGGAUUUAUCGACAA 1371 AD-1230676 ACUGGAUUUAUCGACAAAUGAGG 1372 AD-1230677 UGACGUGGUAGUCACUUGUAAGG 1373 AD-1230678 GCUAGUCACUGGAAAUUGAGGUC 1374 AD-1230679 GGAAAUCAAGGAUGCUCAGUUUA 1375 AD-1230680 GUUCUAUAAAUGAAUGUGCUGAA 1376 AD-1230681 UCUAUAAAUGAAUGUGCUGAAGC 1377 AD-1230682 GUUUUGUUUUGGACUCUCUGUGG 1378 AD-1230683 CAGGCUGGUUUGCAAGAAUGAAA 1379 AD-1230684 ACAGCUAGGACUUAACCUUGAAA 1380 AD-1230685 AAUCCCAUUUGCAGGAUCUGUCU 1381 AD-1230686 GUAAAGUUCAAUUGUGAAAAUGA 1382 AD-1230687 UGUGCACCUCAAAGACUAAGAAA 1383 AD-1230688 UCAAAGACUAAGAAAGCACUGUG 1384 AD-1230689 UUCAGGUGUACUCAUCUCAGAGG 1385 AD-1230690 UUACUCUAGCCAAGGAAUAGUGG 1386 AD-1230691 CACAGUGAUGCCUGUUCUUCAAA 1387 AD-1230692 CAAAAGCAGUGGUUUCUUUACGC 1388 AD-1230693 AAGCAGUGGUUUCUUUACGCUGU 1389 AD-1230694 AGCAGUGGUUUCUUUACGCUGUA 1390 AD-1230695 UACCUAUCAUUACUCGAUGCUGU 1391 AD-1230696 UUAUGACUCCAAGACCAAGAACA 1392 AD-1230697 ACUCCAAGACCAAGAACAAUGAC 1393 AD-1230698 UAUUCACGGACUGGAUUUAUCGA 1394 AD-1230699 GAUGAUUCAGAGGUCACUUCAUU 1395 AD-1230700 AUGAUUCAGAGGUCACUUCAUUU 1396 AD-1230701 UGAUUCAGAGGUCACUUCAUUUU 1397 AD-1230702 CUAGUCACUGGAAAUUGAGGUCC 1398 AD-1230703 AUGUUUCUACACAUUGCUACCUC 1399 AD-1230704 UGUUUCUACACAUUGCUACCUCA 1400 AD-1230705 UUUUGAUGUCUCCAAGUAGUCCA 1401 AD-1230706 ACGUUCUAUAAAUGAAUGUGCUG 1402 AD-1230707 UUUGUUUUGGACUCUCUGUGGUC 1403 AD-1230708 CAUAACCAUGAGCACUACUCUAC 1404 AD-1230709 AAGCAGGCUGGUUUGCAAGAAUG 1405 AD-1230710 UGCAAGAAUGAAAUGAAUGAUUC 1406 AD-1230711 CAAAGACUAAGAAAGCACUGUGC 1407 AD-1230712 UCCUUCAGGUGUACUCAUCUCAG 1408 AD-1230713 CCUUCAGGUGUACUCAUCUCAGA 1409 AD-1230714 AUCCACCAGCUUUAUGAAACUGA 1410 AD-1230715 AAAAGCAGUGGUUUCUUUACGCU 1411 AD-1230716 AAAGCAGUGGUUUCUUUACGCUG 1412 AD-1230717 ACCAAGAACAAUGACAUUGCGCU 1413 AD-1230718 CACGGACUGGAUUUAUCGACAAA 1414 AD-1230719 CUGGAUUUAUCGACAAAUGAGGG 1415 AD-1230720 GGUGACGUGGUAGUCACUUGUAA 1416 AD-1230721 AACGUUCUAUAAAUGAAUGUGCU 1417 AD-1230722 AGAGAAAGAUGUGUUUUGUUUUG 1418 AD-1230723 GAGAAAGAUGUGUUUUGUUUUGG 1419 AD-1230724 AAAGAUGUGUUUUGUUUUGGACU 1420 AD-1230725 GUUUGCAAGAAUGAAAUGAAUGA 1421 AD-1230726 UUUGCAAGAAUGAAAUGAAUGAU 1422 AD-1230727 UUGCAAGAAUGAAAUGAAUGAUU 1423 AD-1230728 GAAAGUCAUGCAAUCCCAUUUGC 1424 AD-1230729 CAAUCCCAUUUGCAGGAUCUGUC 1425 AD-1230730 CCUCUGUAGAGAGCAGCAUUCCC 1426 AD-1230731 UGUAAUGGUGAAAACGUCUUCCU 1427 AD-1230732 UUGUGAAAAUGAAUAUCAUGCAA 1428 AD-1230733 UGUGAAAAUGAAUAUCAUGCAAA 1429 AD-1230734 AUCCUUCAGGUGUACUCAUCUCA 1430 AD-1230735 GACGACUGGAACGAGAACUACGG 1431 AD-1230736 CAGGGACAUGGGCUAUAAGAAUA 1432 AD-1230737 AUUUUUACUCUAGCCAAGGAAUA 1433 AD-1230738 UUUACUCUAGCCAAGGAAUAGUG 1434 AD-1230739 UUUAUGAAACUGAACACAAGUGC 1435 AD-1230740 GAUACCUAUCAUUACUCGAUGCU 1436 AD-1230741 GUAUUCACGGACUGGAUUUAUCG 1437 AD-1230742 AUUCACGGACUGGAUUUAUCGAC 1438 AD-1230743 CUAUGAAAACCAUGGAUACCAAC 1439 AD-1230744 GACGUGGUAGUCACUUGUAAGGG 1440 AD-1230745 CGGCGAAGAAGAGAAAGAUGUGU 1441 AD-1230746 GCGAAGAAGAGAAAGAUGUGUUU 1442 AD-1230747 CGAAGAAGAGAAAGAUGUGUUUU 1443 AD-1230748 GAAGAAGAGAAAGAUGUGUUUUG 1444 AD-1230749 CCAUAACCAUGAGCACUACUCUA 1445 AD-1230750 AGCAGGCUGGUUUGCAAGAAUGA 1446 AD-1230751 AGUCAUGCAAUCCCAUUUGCAGG 1447 AD-1230752 AUCCCAUUUGCAGGAUCUGUCUG 1448 AD-1230753 UACUCAUCUCAGAGGAAGUCCUG 1449 AD-1230754 AAGACGACUGGAACGAGAACUAC 1450 AD-1230755 CUCUAGCCAAGGAAUAGUGGAUG 1451 AD-1230756 AAAAACUGUACCACAGUGAUGCC 1452 AD-1230757 ACCACAGUGAUGCCUGUUCUUCA 1453 AD-1230758 AGAACAAUGACAUUGCGCUGAUG 1454 AD-1230759 AACAAUGACAUUGCGCUGAUGAA 1455 AD-1230760 AGAAAGAUGUGUUUUGUUUUGGA 1456 AD-1230761 AUUGCCAAGUGCCAUAACCAUGA 1457 AD-1230762 AGUGUGCACCUCAAAGACUAAGA 1458 AD-1230763 AAAGACUAAGAAAGCACUGUGCA 1459 AD-1230764 UCAGGUGUACUCAUCUCAGAGGA 1460 AD-1230765 UACCACAGUGAUGCCUGUUCUUC 1461 AD-1230766 CCACAGUGAUGCCUGUUCUUCAA 1462 AD-1230767 CCUGUUCUUCAAAAGCAGUGGUU 1463 AD-1230768 CAAGAACAAUGACAUUGCGCUGA 1464 AD-1230769 GCAAGAUGGCUUUGAACUCAGGG 1465 AD-1230770 AAGAUGGCUUUGAACUCAGGGUC 1466 AD-1230771 UGGCACUCUCUGCCAUUCUGUGC 1467 AD-1230772 GUGGUGACGUGGUAGUCACUUGU 1468 AD-1230773 UGGUGACGUGGUAGUCACUUGUA 1469 AD-1230774 UGCUAGUCACUGGAAAUUGAGGU 1470 AD-1230775 UCACUGGAAAUUGAGGUCCAUGG 1471 AD-1230776 AAGAAGAGAAAGAUGUGUUUUGU 1472 AD-1230777 AAGAGAAAGAUGUGUUUUGUUUU 1473 AD-1230778 GAAAGAUGUGUUUUGUUUUGGAC 1474 AD-1230779 UUUUGUUUUGGACUCUCUGUGGU 1475 AD-1230780 GGUGUACUCAUCUCAGAGGAAGU 1476 AD-1230781 GACCAAGAACAAUGACAUUGCGC 1477 AD-1230782 AAGAACAAUGACAUUGCGCUGAU 1478 AD-1230783 AAUGACAUUGCGCUGAUGAAGCU 1479 AD-1230784 GAGGAGAAAGGGAAGACCUCAGA 1480 AD-1230785 CUUUGGCACUCUCUGCCAUUCUG 1481 AD-1230786 GGCGAAGAAGAGAAAGAUGUGUU 1482 AD-1230787 AGAAGAGAAAGAUGUGUUUUGUU 1483 AD-1230788 GCAGGCUGGUUUGCAAGAAUGAA 1484 AD-1230789 CAUGCAAUCCCAUUUGCAGGAUC 1485 AD-1230790 AUGCAAUCCCAUUUGCAGGAUCU 1486 AD-1230791 CACAUGCCUCUGUAGAGAGCAGC 1487 AD-1230792 ACUCUGGGAUAGAGUGCGACUCC 1488 AD-1230793 GUACUCAUCUCAGAGGAAGUCCU 1489 AD-1230794 AGACGACUGGAACGAGAACUACG 1490 AD-1230795 AAUUUUUACUCUAGCCAAGGAAU 1491 AD-1230796 UUUUACUCUAGCCAAGGAAUAGU 1492 AD-1230797 AAACUGUACCACAGUGAUGCCUG 1493 AD-1230798 GUACCACAGUGAUGCCUGUUCUU 1494 AD-1230799 GAACAAUGACAUUGCGCUGAUGA 1495 AD-1230800 CAAUGACAUUGCGCUGAUGAAGC 1496 AD-1230801 GACCAGGAGUGUACGGGAAUGUG 1497 AD-1230802 GGUAUUCACGGACUGGAUUUAUC 1498 AD-1230803 UGGAUUUAUCGACAAAUGAGGGC 1499 AD-1230804 UUAGAGAUGAUUCAGAGGUCACU 1500 AD-1230805 GAAGAGAAAGAUGUGUUUUGUUU 1501 AD-1230806 GCCAAGUGCCAUAACCAUGAGCA 1502 AD-1230807 GCCAUAACCAUGAGCACUACUCU 1503 AD-1230808 CCCAUUUGCAGGAUCUGUCUGUG 1504 AD-1230809 ACUCAUCUCAGAGGAAGUCCUGG 1505 AD-1230810 ACCAGGAGUGUACGGGAAUGUGA 1506 AD-1230811 UGUCUGGCUUUGGCACUCUCUGC 1507 AD-1230812 UUUGGCACUCUCUGCCAUUCUGU 1508 AD-1230813 UUGGCACUCUCUGCCAUUCUGUG 1509 AD-1230814 AAACCAUGGAUACCAACCGGAAA 1510 AD-1230815 AACCAUGGAUACCAACCGGAAAA 1511 AD-1230816 GGGGGAAAUCAAGGAUGCUCAGU 1512 AD-1230817 UGCCAAGUGCCAUAACCAUGAGC 1513 AD-1230818 GUCAUGCAAUCCCAUUUGCAGGA 1514 AD-1230819 UCCCAUUUGCAGGAUCUGUCUGU 1515 AD-1230820 UUUGCAGGAUCUGUCUGUGCACA 1516

TABLE 4 TMPRSS2 Single Dose Screens in PHH cells 10 nM 1 nM 0.1 nM Duplex ID Avg SD Avg SD Avg SD AD-1230667.1 20.4 5.1 32.9 12.3 42.5 11.3 AD-1230624.1 14.7 5.8 24.3 4.8 22.1 1.5 AD-1230625.1 14.3 1.1 19.6 3.5 23.2 10.1 AD-1230595.1 17.3 4.8 22.3 4.5 31.9 1.7 AD-1230547.1 17.6 3.0 27.4 6.8 41.0 5.2 AD-1230526.1 7.9 2.0 17.0 2.6 22.7 6.9 AD-1230527.1 6.6 1.2 13.4 1.1 18.6 3.8 AD-1230548.1 17.2 4.7 19.7 3.0 41.6 11.0 AD-1230528.1 10.5 2.3 30.1 9.0 23.0 6.2 AD-1230549.1 20.4 6.6 20.4 6.4 36.1 12.3 AD-1230529.1 18.3 2.4 34.9 5.2 39.0 3.9 AD-1230641.1 33.3 8.1 67.4 9.3 52.9 7.4 AD-1230740.1 68.6 3.3 74.5 8.3 85.9 9.3 AD-1230596.1 15.2 4.6 26.3 8.0 41.5 6.7 AD-1230695.1 14.9 3.8 18.4 4.4 40.6 6.7 AD-1230567.1 30.6 2.3 53.5 15.9 58.3 22.4 AD-1230642.1 49.5 23.9 75.1 17.2 82.4 12.0 AD-1230626.1 15.8 0.7 18.7 5.9 35.7 6.8 AD-1230598.1 30.1 1.2 42.9 9.9 59.1 5.3 AD-1230670.1 26.6 2.6 22.9 3.7 31.6 11.8 AD-1230599.1 19.3 4.1 30.8 4.2 58.1 11.5 AD-1230601.1 38.5 6.3 56.4 12.8 52.5 8.7 AD-1230602.1 16.3 4.4 22.0 2.8 31.9 10.7 AD-1230673.1 20.3 3.2 34.8 15.4 49.1 10.3 AD-1230630.1 25.9 5.9 32.5 9.2 40.4 6.6 AD-1230769.1 57.4 2.0 71.0 14.9 82.6 7.1 AD-1230770.1 47.1 3.1 63.5 7.4 65.0 5.5 AD-1230743.1 49.9 6.6 46.1 2.5 71.7 10.1 AD-1230814.1 60.8 12.2 61.9 4.2 49.9 5.3 AD-1230815.1 69.4 11.6 103.0 31.5 85.2 12.4 AD-1230762.1 53.5 6.4 59.0 5.2 64.3 4.5 AD-1230663.1 25.8 1.7 23.6 4.9 43.7 15.7 AD-1230687.1 45.8 4.6 65.5 18.8 82.5 8.0 AD-1230588.1 30.8 3.0 24.4 11.7 61.1 6.9 AD-1230589.1 26.6 4.9 53.2 3.8 48.6 14.3 AD-1230617.1 23.3 3.3 29.2 8.5 27.4 5.1 AD-1230618.1 26.5 5.2 27.9 8.2 32.1 13.1 AD-1230619.1 35.7 5.7 53.2 11.2 51.6 8.6 AD-1230688.1 20.3 7.5 25.6 2.7 44.9 11.4 AD-1230711.1 27.3 8.0 25.3 5.1 43.8 5.1 AD-1230763.1 70.8 14.5 83.8 29.2 80.8 13.0 AD-1230792.1 87.2 14.8 50.0 7.0 101.8 16.8 AD-1230734.1 34.1 2.7 30.6 5.2 56.0 10.8 AD-1230712.1 17.9 1.0 19.5 5.1 31.5 14.6 AD-1230713.1 21.3 3.9 24.1 5.7 34.6 6.2 AD-1230689.1 24.0 7.4 34.3 14.1 50.7 19.5 AD-1230764.1 39.6 11.8 53.7 12.1 60.2 4.6 AD-1230780.1 37.0 5.6 41.5 11.3 71.4 7.3 AD-1230541.1 45.2 11.7 45.3 13.6 60.5 11.3 AD-1230793.1 91.9 11.0 54.2 13.3 103.6 22.4 AD-1230753.1 105.5 55.5 74.1 22.2 112.8 36.1 AD-1230809.1 71.3 10.6 70.9 2.2 76.4 16.5 AD-1230754.1 32.0 3.8 50.0 11.6 83.2 29.9 AD-1230794.1 48.7 5.3 37.4 3.2 65.7 11.6 AD-1230735.1 41.9 6.1 38.6 3.4 61.5 6.7 AD-1230736.1 36.5 6.0 42.1 5.5 67.3 12.2 AD-1230795.1 50.1 3.8 38.6 6.8 48.2 5.5 AD-1230737.1 37.5 8.2 38.3 8.3 47.9 17.0 AD-1230796.1 56.4 8.9 42.4 2.1 61.0 18.5 AD-1230738.1 37.4 8.1 32.6 4.3 68.6 25.9 AD-1230690.1 63.0 12.5 53.0 7.3 73.9 13.2 AD-1230755.1 42.9 7.9 64.7 9.0 74.0 15.0 AD-1230590.1 17.9 3.9 27.8 7.6 52.6 4.1 AD-1230664.1 44.5 8.6 77.8 12.0 102.7 15.0 AD-1230714.1 38.9 4.0 46.4 8.9 50.8 6.9 AD-1230620.1 19.0 6.8 39.1 7.3 41.8 6.6 AD-1230665.1 29.9 9.4 49.9 6.5 64.0 13.7 AD-1230739.1 79.0 12.8 67.8 7.2 108.0 21.6 AD-1230666.1 24.6 8.3 43.6 8.7 60.0 6.5 AD-1230621.1 36.0 4.4 64.8 6.5 62.9 17.3 AD-1230542.1 9.4 3.4 24.3 3.3 35.9 3.7 AD-1230543.1 8.4 3.6 12.9 4.0 21.7 6.6 AD-1230544.1 23.8 1.6 56.5 5.4 58.6 8.9 AD-1230545.1 12.5 2.9 36.2 11.1 40.2 15.1 AD-1230756.1 32.4 3.6 45.9 4.9 70.7 6.3 AD-1230797.1 46.8 5.3 42.6 8.4 62.7 12.1 AD-1230798.1 28.1 1.6 41.1 9.7 57.2 14.0 AD-1230765.1 84.0 7.8 99.2 11.1 75.7 12.5 AD-1230757.1 17.6 7.6 19.1 2.3 24.3 6.4 AD-1230766.1 16.0 1.1 22.6 7.7 33.7 6.2 AD-1230691.1 16.1 3.9 23.9 2.5 40.5 6.9 AD-1230636.1 26.9 5.6 39.8 13.5 47.4 9.6 AD-1230591.1 36.6 4.3 49.9 8.3 66.1 14.8 AD-1230592.1 23.5 3.9 41.3 8.5 60.1 12.1 AD-1230564.1 16.7 5.2 33.0 14.1 52.1 11.3 AD-1230593.1 26.3 6.4 33.3 6.6 63.3 10.2 AD-1230565.1 27.5 6.4 40.6 16.9 73.9 4.9 AD-1230767.1 32.3 4.6 35.1 1.9 50.9 5.4 AD-1230622.1 54.6 12.5 77.6 25.6 78.5 21.4 AD-1230637.1 40.2 8.6 54.2 18.4 67.8 8.7 AD-1230594.1 23.3 3.7 35.8 12.1 49.5 11.7 AD-1230623.1 29.3 12.7 42.6 8.0 48.7 6.9 AD-1230566.1 24.6 5.0 43.7 8.7 61.3 13.3 AD-1230638.1 35.2 6.4 42.8 6.8 57.5 20.0 AD-1230639.1 14.9 3.5 26.1 8.0 44.8 8.5 AD-1230668.1 21.1 7.3 28.3 5.0 40.2 7.3 AD-1230692.1 12.6 1.5 16.3 3.1 28.3 1.8 AD-1230715.1 32.6 4.2 24.1 1.9 37.8 1.9 AD-1230716.1 22.1 3.7 22.6 4.7 43.4 11.5 AD-1230693.1 18.6 4.7 22.2 4.4 42.2 11.3 AD-1230694.1 18.6 2.0 23.9 6.6 36.5 10.1 AD-1230546.1 47.5 6.0 76.7 15.1 95.8 8.5 AD-1230525.1 16.7 4.5 24.0 5.3 33.2 6.1 AD-1230640.1 31.8 7.7 50.1 14.4 88.4 27.9 AD-1230669.1 27.3 6.9 35.4 3.8 56.3 7.5 AD-1230568.1 26.2 3.7 48.2 4.6 51.9 15.8 AD-1230597.1 20.0 3.1 43.0 7.7 44.5 9.0 AD-1230530.1 17.8 0.9 43.0 9.3 52.3 11.0 AD-1230569.1 13.0 2.1 20.1 5.7 32.8 12.5 AD-1230570.1 11.6 1.9 23.7 5.9 30.7 9.5 AD-1230627.1 15.5 2.4 35.6 12.6 34.5 4.6 AD-1230571.1 18.0 4.6 15.9 5.0 36.3 5.1 AD-1230572.1 11.1 4.5 21.8 6.1 32.5 13.7 AD-1230643.1 26.2 3.2 36.3 11.5 43.4 9.3 AD-1230573.1 11.3 5.7 21.8 3.5 27.4 9.8 AD-1230531.1 18.0 4.2 38.8 13.0 48.4 7.2 AD-1230550.1 13.7 1.4 19.6 9.2 34.5 5.3 AD-1230551.1 33.8 4.4 48.6 10.7 57.9 7.1 AD-1230574.1 27.9 5.0 53.3 8.8 64.2 9.6 AD-1230552.1 21.8 4.8 41.0 11.0 53.9 10.6 AD-1230553.1 26.2 7.3 46.1 3.5 63.4 10.5 AD-1230554.1 53.6 3.4 71.6 11.1 85.8 14.1 AD-1230533.1 35.8 5.8 53.7 8.2 70.6 9.7 AD-1230696.1 40.0 2.8 48.2 16.9 60.1 6.6 AD-1230644.1 65.8 14.7 51.9 21.4 83.9 10.3 AD-1230628.1 22.8 5.8 57.2 13.4 55.1 16.5 AD-1230697.1 25.1 5.0 30.9 8.0 60.9 16.4 AD-1230645.1 26.6 9.1 29.5 2.3 52.4 9.5 AD-1230629.1 27.9 5.7 40.3 7.7 49.5 8.7 AD-1230646.1 41.8 14.3 40.5 13.3 49.7 14.7 AD-1230671.1 15.9 1.0 31.1 10.5 61.6 19.8 AD-1230672.1 14.3 2.0 30.6 6.2 38.7 8.7 AD-1230781.1 28.8 3.8 60.4 38.0 94.0 14.7 AD-1230717.1 83.6 13.2 73.4 8.4 91.1 15.0 AD-1230600.1 23.3 4.9 31.6 12.4 48.3 10.5 AD-1230768.1 70.8 13.3 76.8 11.7 99.9 56.4 AD-1230782.1 16.9 4.2 26.6 10.5 56.6 6.9 AD-1230758.1 26.7 4.9 29.3 4.6 38.4 8.4 AD-1230799.1 135.0 27.3 104.5 26.6 94.7 32.7 AD-1230759.1 23.2 4.0 25.2 5.4 54.2 10.5 AD-1230800.1 60.6 8.3 67.5 32.3 84.5 16.5 AD-1230783.1 20.0 3.6 27.9 6.4 48.4 7.7 AD-1230575.1 29.7 6.5 67.3 4.7 79.0 15.3 AD-1230784.1 45.4 3.6 51.9 8.3 81.3 24.3 AD-1230801.1 114.4 16.0 109.3 22.2 89.1 25.4 AD-1230810.1 73.0 23.5 43.6 3.1 68.5 12.6 AD-1230802.1 29.2 6.4 31.1 1.2 44.6 4.0 AD-1230741.1 24.2 4.6 30.9 11.9 50.5 13.7 AD-1230698.1 20.0 2.9 18.8 1.9 37.2 7.0 AD-1230742.1 73.0 3.9 78.8 5.7 80.2 13.9 AD-1230674.1 17.8 5.4 24.4 4.7 43.2 2.2 AD-1230675.1 27.3 4.6 33.2 4.3 52.3 7.0 AD-1230718.1 31.3 4.6 32.2 6.2 59.0 6.1 AD-1230534.1 20.4 4.8 27.8 6.8 45.5 8.9 AD-1230647.1 40.2 12.8 38.3 4.5 70.3 10.8 AD-1230676.1 38.0 6.3 35.1 3.4 64.5 10.4 AD-1230719.1 43.6 6.0 35.3 6.1 63.6 2.5 AD-1230804.1 15.4 1.6 22.7 6.1 22.0 4.2 AD-1230699.1 11.0 1.5 12.0 2.2 23.1 7.2 AD-1230700.1 9.0 2.9 8.5 3.4 19.5 4.3 AD-1230701.1 30.4 6.5 28.4 4.6 54.0 20.5 AD-1230648.1 16.2 0.5 25.9 10.4 32.9 7.9 AD-1230603.1 12.5 0.5 15.8 2.4 25.2 9.8 AD-1230604.1 9.9 2.6 22.2 0.6 28.6 7.9 AD-1230649.1 27.7 3.3 53.5 15.3 51.8 4.4 AD-1230650.1 21.0 3.6 40.5 18.2 43.7 7.5 AD-1230555.1 14.5 6.0 17.3 4.2 32.4 10.6 AD-1230811.1 45.8 6.6 35.1 2.6 65.3 13.0 AD-1230785.1 36.3 3.7 37.5 8.2 55.4 16.3 AD-1230812.1 44.4 4.4 38.0 2.3 53.7 5.2 AD-1230813.1 31.0 2.1 36.8 13.1 51.7 10.9 AD-1230771.1 50.0 0.8 51.4 7.6 68.8 9.8 AD-1230772.1 56.2 10.3 63.5 13.8 69.3 8.1 AD-1230773.1 56.9 5.9 56.0 11.9 73.6 11.9 AD-1230720.1 42.1 7.8 36.1 5.6 49.4 3.5 AD-1230631.1 26.8 6.7 32.3 5.7 39.1 10.3 AD-1230677.1 37.9 6.6 32.3 3.9 42.7 5.9 AD-1230744.1 48.7 6.8 58.5 17.2 64.4 5.7 AD-1230651.1 57.5 4.3 58.6 8.5 71.2 17.0 AD-1230652.1 45.1 10.1 48.8 18.3 67.3 5.8 AD-1230576.1 42.7 6.7 72.2 7.7 65.7 10.3 AD-1230774.1 45.5 1.6 48.0 9.4 65.4 8.2 AD-1230678.1 23.5 3.0 29.1 6.2 29.1 5.0 AD-1230702.1 45.2 3.1 37.3 3.2 65.4 6.3 AD-1230775.1 43.9 16.6 51.8 20.5 63.1 10.0 AD-1230816.1 75.2 2.8 83.1 12.7 67.3 12.7 AD-1230679.1 39.8 4.0 76.1 13.0 83.0 24.4 AD-1230605.1 19.6 6.1 39.5 7.5 45.5 4.7 AD-1230632.1 21.0 5.3 30.3 3.7 42.3 6.9 AD-1230653.1 44.6 11.0 43.8 6.8 55.8 17.0 AD-1230606.1 20.4 6.0 32.7 7.0 40.4 7.8 AD-1230556.1 35.7 4.3 39.7 5.5 53.4 4.8 AD-1230703.1 40.8 6.1 36.6 16.1 58.4 10.0 AD-1230704.1 29.5 4.9 23.9 5.8 35.8 7.3 AD-1230654.1 25.0 4.4 36.9 8.5 51.5 9.1 AD-1230577.1 41.3 7.2 54.8 12.7 68.2 11.1 AD-1230535.1 23.4 7.8 34.2 4.6 33.8 5.5 AD-1230705.1 27.1 3.5 29.7 8.2 49.1 2.7 AD-1230655.1 48.7 8.0 28.5 7.6 44.4 12.6 AD-1230521.1 34.8 7.8 53.0 5.0 52.1 9.9 AD-1230557.1 26.1 5.2 37.4 10.2 45.5 8.7 AD-1230607.1 23.8 5.0 37.3 19.4 48.3 11.6 AD-1230721.1 28.8 6.6 24.5 5.9 25.2 3.1 AD-1230706.1 19.4 5.4 20.0 6.0 35.9 4.9 AD-1230656.1 43.4 5.6 68.7 4.7 65.9 8.2 AD-1230680.1 22.4 3.4 46.2 16.3 53.3 5.5 AD-1230681.1 31.2 9.1 33.9 2.8 52.5 6.1 AD-1230745.1 31.5 7.9 26.8 5.1 36.0 11.5 AD-1230786.1 33.8 6.2 36.9 5.1 53.0 2.8 AD-1230746.1 40.2 7.4 50.9 14.3 76.9 28.7 AD-1230747.1 46.6 4.5 47.2 5.2 61.8 6.0 AD-1230748.1 51.4 15.5 56.6 20.1 62.8 7.0 AD-1230776.1 27.4 4.4 38.1 8.2 47.9 11.9 AD-1230787.1 36.8 7.2 38.9 7.2 59.5 11.5 AD-1230805.1 38.6 6.5 32.4 7.0 53.0 10.1 AD-1230777.1 39.0 5.1 51.6 6.1 64.6 7.2 AD-1230722.1 31.7 4.0 25.7 3.9 29.2 1.4 AD-1230723.1 26.5 4.6 23.7 3.6 46.1 14.9 AD-1230760.1 37.5 12.2 37.4 4.9 40.7 7.4 AD-1230778.1 52.6 10.0 52.0 11.6 68.0 15.8 AD-1230724.1 55.8 6.6 54.9 15.1 93.2 23.2 AD-1230578.1 42.7 2.8 48.6 8.8 76.7 4.8 AD-1230608.1 32.6 3.0 41.1 6.6 62.2 7.5 AD-1230579.1 43.8 7.1 53.7 9.4 73.7 5.3 AD-1230682.1 35.2 5.4 47.2 12.6 67.5 11.6 AD-1230779.1 63.7 5.3 72.5 8.0 98.2 10.3 AD-1230707.1 37.7 0.7 38.9 3.7 48.8 3.0 AD-1230761.1 49.2 21.2 59.1 15.5 83.4 25.8 AD-1230817.1 100.9 11.0 73.1 9.0 79.5 9.0 AD-1230806.1 51.8 9.8 59.0 8.5 93.2 29.6 AD-1230807.1 62.8 7.3 56.2 21.6 67.2 25.0 AD-1230749.1 65.6 2.7 57.9 4.4 54.5 10.5 AD-1230708.1 23.0 2.8 24.2 1.2 43.4 15.1 AD-1230709.1 50.9 3.5 45.8 15.2 89.5 17.7 AD-1230750.1 34.7 10.8 31.9 8.4 44.0 7.6 AD-1230788.1 36.5 9.0 39.2 4.0 47.7 4.7 AD-1230683.1 27.3 3.2 34.7 5.4 48.0 11.9 AD-1230580.1 31.8 2.7 38.8 10.2 47.6 13.2 AD-1230581.1 38.0 3.9 59.3 11.8 66.2 10.4 AD-1230582.1 26.9 3.9 48.9 9.2 51.2 10.1 AD-1230609.1 23.7 7.4 21.7 6.0 39.1 6.0 AD-1230633.1 21.8 6.2 29.3 6.3 48.2 20.0 AD-1230725.1 47.1 8.2 42.0 5.4 49.5 6.9 AD-1230726.1 41.8 8.2 40.5 7.0 50.5 6.7 AD-1230727.1 43.9 8.9 37.8 7.0 52.1 6.0 AD-1230710.1 33.5 4.3 33.7 4.6 57.6 12.1 AD-1230610.1 19.7 3.8 26.6 3.7 40.7 9.1 AD-1230657.1 48.2 8.6 67.5 6.1 76.4 5.1 AD-1230658.1 40.1 13.3 52.5 7.6 59.7 9.6 AD-1230684.1 28.7 9.5 33.3 6.9 41.4 6.9 AD-1230611.1 37.8 7.2 69.9 10.0 54.5 7.7 AD-1230558.1 27.3 6.5 29.5 10.8 43.3 2.8 AD-1230583.1 39.0 6.7 63.0 7.7 90.0 15.8 AD-1230634.1 41.9 5.9 81.4 21.8 74.3 21.4 AD-1230659.1 32.9 5.8 28.0 7.8 51.4 5.7 AD-1230660.1 38.8 8.7 42.7 10.6 55.1 11.8 AD-1230728.1 51.8 6.3 44.3 3.9 52.3 6.2 AD-1230661.1 49.5 7.4 52.0 9.9 66.8 12.1 AD-1230662.1 48.2 6.3 45.5 7.6 61.1 12.1 AD-1230751.1 43.5 19.3 47.1 15.8 72.7 10.9 AD-1230818.1 72.1 12.7 67.1 3.8 67.1 7.9 AD-1230789.1 44.9 10.6 63.0 5.9 59.1 14.8 AD-1230790.1 40.1 7.5 43.9 6.2 56.0 11.3 AD-1230729.1 50.0 5.3 37.5 7.7 54.5 5.2 AD-1230685.1 37.7 9.5 33.0 3.9 59.2 5.6 AD-1230752.1 50.1 13.3 54.0 16.9 57.7 24.6 AD-1230819.1 64.0 15.5 50.1 5.2 72.7 14.3 AD-1230808.1 43.9 2.5 58.1 15.7 54.2 10.5 AD-1230820.1 48.9 10.8 45.4 12.6 54.9 7.9 AD-1230791.1 99.4 16.3 75.7 21.7 89.2 19.1 AD-1230730.1 36.9 7.3 33.2 12.0 48.4 9.2 AD-1230536.1 31.2 7.1 37.7 10.5 53.6 14.2 AD-1230612.1 34.5 10.3 43.8 3.6 47.8 14.6 AD-1230560.1 51.5 15.0 59.7 9.7 67.0 12.2 AD-1230613.1 33.1 9.9 38.3 9.5 41.9 5.8 AD-1230537.1 28.3 2.5 48.1 2.8 52.3 3.1 AD-1230731.1 83.9 55.9 47.1 8.9 84.8 21.2 AD-1230614.1 29.6 9.0 33.9 6.1 36.4 12.4 AD-1230524.1 15.0 3.9 29.9 6.4 36.6 3.8 AD-1230584.1 28.2 4.4 49.5 10.9 52.7 12.7 AD-1230562.1 31.3 4.5 57.1 13.3 55.9 18.1 AD-1230538.1 31.6 11.5 52.2 7.5 52.6 4.6 AD-1230539.1 27.1 1.1 40.6 2.6 43.7 10.6 AD-1230563.1 34.3 7.9 43.4 14.7 54.8 4.6 AD-1230522.1 24.0 7.0 40.4 7.3 38.9 5.6 AD-1230523.1 24.5 4.8 33.2 13.4 35.5 8.2 AD-1230585.1 35.9 3.9 41.6 2.9 66.7 5.4 AD-1230586.1 31.1 4.0 42.0 3.8 51.5 10.0 AD-1230540.1 28.6 2.9 38.7 12.1 48.1 1.0 AD-1230615.1 23.5 5.0 35.5 13.1 31.7 11.2 AD-1230587.1 26.9 6.3 25.6 5.3 42.0 8.7 AD-1230616.1 27.5 3.8 33.8 9.7 28.7 7.5 AD-1230686.1 18.4 1.5 17.4 6.5 35.5 10.0 AD-1230635.1 42.1 10.1 64.4 12.0 74.0 14.7 AD-1230732.1 40.1 6.9 40.7 3.6 59.4 7.7 AD-1230733.1 45.4 4.6 42.2 7.2 52.1 5.6

Example 3 In Vivo Screening of dsRNA Duplexes in Mice

siRNA molecules targeting the TMPRSS2 gene, identified from the above in vitro studies, are evaluated in vivo.

Mice previously infected with a coronavirus, e.g., severe acute respiratory syndrome-2 (SARS-2)-CoV-2, are administered, via pulmonary or subcutaneous delivery, a dsRNA molecule at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg. Uptake of dsRNA in bronchioles and alveoli and expression level of target gene in whole lung of treated mice are measured. Expression levels of coronavirus target genes are further evaluated by in situ hybridization in mice bronchus and bronchiole.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

INFORMAL SEQUENCE LISTING <210>    1 <211> 3450 <212> DNA <213> Homo sapiens <400>    1 gagtaggcgc gagctaagca ggaggcggag gcggaggcgg agggcgaggg gcggggagcg 60 ccgcctggag cgcggcaggt catattgaac attccagata cctatcatta ctcgatgctg 120 ttgataacag caagatggct ttgaactcag ggtcaccacc agctattgga ccttactatg 180 aaaaccatgg ataccaaccg gaaaacccct atcccgcaca gcccactgtg gtccccactg 240 tctacgaggt gcatccggct cagtactacc cgtcccccgt gccccagtac gccccgaggg 300 tcctgacgca ggcttccaac cccgtcgtct gcacgcagcc caaatcccca tccgggacag 360 tgtgcacctc aaagactaag aaagcactgt gcatcacctt gaccctgggg accttcctcg 420 tgggagctgc gctggccgct ggcctactct ggaagttcat gggcagcaag tgctccaact 480 ctgggataga gtgcgactcc tcaggtacct gcatcaaccc ctctaactgg tgtgatggcg 540 tgtcacactg ccccggcggg gaggacgaga atcggtgtgt tcgcctctac ggaccaaact 600 tcatccttca ggtgtactca tctcagagga agtcctggca ccctgtgtgc caagacgact 660 ggaacgagaa ctacgggcgg gcggcctgca gggacatggg ctataagaat aatttttact 720 ctagccaagg aatagtggat gacagcggat ccaccagctt tatgaaactg aacacaagtg 780 ccggcaatgt cgatatctat aaaaaactgt accacagtga tgcctgttct tcaaaagcag 840 tggtttcttt acgctgtata gcctgcgggg tcaacttgaa ctcaagccgc cagagcagga 900 ttgtgggcgg cgagagcgcg ctcccggggg cctggccctg gcaggtcagc ctgcacgtcc 960 agaacgtcca cgtgtgcgga ggctccatca tcacccccga gtggatcgtg acagccgccc 1020 actgcgtgga aaaacctctt aacaatccat ggcattggac ggcatttgcg gggattttga 1080 gacaatcttt catgttctat ggagccggat accaagtaga aaaagtgatt tctcatccaa 1140 attatgactc caagaccaag aacaatgaca ttgcgctgat gaagctgcag aagcctctga 1200 ctttcaacga cctagtgaaa ccagtgtgtc tgcccaaccc aggcatgatg ctgcagccag 1260 aacagctctg ctggatttcc gggtgggggg ccaccgagga gaaagggaag acctcagaag 1320 tgctgaacgc tgccaaggtg cttctcattg agacacagag atgcaacagc agatatgtct 1380 atgacaacct gatcacacca gccatgatct gtgccggctt cctgcagggg aacgtcgatt 1440 cttgccaggg tgacagtgga gggcctctgg tcacttcgaa gaacaatatc tggtggctga 1500 taggggatac aagctggggt tctggctgtg ccaaagctta cagaccagga gtgtacggga 1560 atgtgatggt attcacggac tggatttatc gacaaatgag ggcagacggc taatccacat 1620 ggtcttcgtc cttgacgtcg ttttacaaga aaacaatggg gctggttttg cttccccgtg 1680 catgatttac tcttagagat gattcagagg tcacttcatt tttattaaac agtgaacttg 1740 tctggctttg gcactctctg ccattctgtg caggctgcag tggctcccct gcccagcctg 1800 ctctccctaa ccccttgtcc gcaaggggtg atggccggct ggttgtgggc actggcggtc 1860 aagtgtggag gagaggggtg gaggctgccc cattgagatc ttcctgctga gtcctttcca 1920 ggggccaatt ttggatgagc atggagctgt cacctctcag ctgctggatg acttgagatg 1980 aaaaaggaga gacatggaaa gggagacagc caggtggcac ctgcagcggc tgccctctgg 2040 ggccacttgg tagtgtcccc agcctacctc tccacaaggg gattttgctg atgggttctt 2100 agagccttag cagccctgga tggtggccag aaataaaggg accagccctt catgggtggt 2160 gacgtggtag tcacttgtaa ggggaacaga aacatttttg ttcttatggg gtgagaatat 2220 agacagtgcc cttggtgcga gggaagcaat tgaaaaggaa cttgccctga gcactcctgg 2280 tgcaggtctc cacctgcaca ttgggtgggg ctcctgggag ggagactcag ccttcctcct 2340 catcctccct gaccctgctc ctagcaccct ggagagtgca catgcccctt ggtcctggca 2400 gggcgccaag tctggcacca tgttggcctc ttcaggcctg ctagtcactg gaaattgagg 2460 tccatggggg aaatcaagga tgctcagttt aaggtacact gtttccatgt tatgtttcta 2520 cacattgcta cctcagtgct cctggaaact tagcttttga tgtctccaag tagtccacct 2580 tcatttaact ctttgaaact gtatcatctt tgccaagtaa gagtggtggc ctatttcagc 2640 tgctttgaca aaatgactgg ctcctgactt aacgttctat aaatgaatgt gctgaagcaa 2700 agtgcccatg gtggcggcga agaagagaaa gatgtgtttt gttttggact ctctgtggtc 2760 ccttccaatg ctgtgggttt ccaaccaggg gaagggtccc ttttgcattg ccaagtgcca 2820 taaccatgag cactactcta ccatggttct gcctcctggc caagcaggct ggtttgcaag 2880 aatgaaatga atgattctac agctaggact taaccttgaa atggaaagtc atgcaatccc 2940 atttgcagga tctgtctgtg cacatgcctc tgtagagagc agcattccca gggaccttgg 3000 aaacagttgg cactgtaagg tgcttgctcc ccaagacaca tcctaaaagg tgttgtaatg 3060 gtgaaaacgt cttccttctt tattgcccct tcttatttat gtgaacaact gtttgtcttt 3120 ttttgtatct tttttaaact gtaaagttca attgtgaaaa tgaatatcat gcaaataaat 3180 tatgcaattt ttttttcaaa gtaactactg catctttgaa gttctgcctg gtgagtagga 3240 ccagcctcca tttccttata agggggtgat gttgaggctg ctggtcagag gaccaaaggt 3300 gaggcaaggc cagacttggt gctcctgtgg ttggtgccct cagttcctgc agcctgtcct 3360 gttggagagg tccctcaaat gactccttct tattattcta ttagtctgtt tccatgctcc 3420 taataaagac atacccaaga ctgcaattta 3450 <210>    2 <211> 3250 <212> DNA <213> Homo sapiens <400>    2 accagggtcc cggctcgggg tccgggctgg ggaggggaac ctgggcgcct gggacccgcc 60 gatgccccct gccccgcccg gaggtgaaag cgggtgtgag gagcgcggcg cggcaggtca 120 tattgaacat tccagatacc tatcattact cgatgctgtt gataacagca agatggcttt 180 gaactcaggg tcaccaccag ctattggacc ttactatgaa aaccatggat accaaccgga 240 aaacccctat cccgcacagc ccactgtggt ccccactgtc tacgaggtgc atccggctca 300 gtactacccg tcccccgtgc cccagtacgc cccgagggtc ctgacgcagg cttccaaccc 360 cgtcgtctgc acgcagccca aatccccatc cgggacagtg tgcacctcaa agactaagaa 420 agcactgtgc atcaccttga ccctggggac cttcctcgtg ggagctgcgc tggccgctgg 480 cctactctgg aagttcatgg gcagcaagtg ctccaactct gggatagagt gcgactcctc 540 aggtacctgc atcaacccct ctaactggtg tgatggcgtg tcacactgcc ccggcgggga 600 ggacgagaat cggtgtgttc gcctctacgg accaaacttc atccttcagg tgtactcatc 660 tcagaggaag tcctggcacc ctgtgtgcca agacgactgg aacgagaact acgggcgggc 720 ggcctgcagg gacatgggct ataagaataa tttttactct agccaaggaa tagtggatga 780 cagcggatcc accagcttta tgaaactgaa cacaagtgcc ggcaatgtcg atatctataa 840 aaaactgtac cacagtgatg cctgttcttc aaaagcagtg gtttctttac gctgtatagc 900 ctgcggggtc aacttgaact caagccgcca gagcaggatt gtgggcggcg agagcgcgct 960 cccgggggcc tggccctggc aggtcagcct gcacgtccag aacgtccacg tgtgcggagg 1020 ctccatcatc acccccgagt ggatcgtgac agccgcccac tgcgtggaaa aacctcttaa 1080 caatccatgg cattggacgg catttgcggg gattttgaga caatctttca tgttctatgg 1140 agccggatac caagtagaaa aagtgatttc tcatccaaat tatgactcca agaccaagaa 1200 caatgacatt gcgctgatga agctgcagaa gcctctgact ttcaacgacc tagtgaaacc 1260 agtgtgtctg cccaacccag gcatgatgct gcagccagaa cagctctgct ggatttccgg 1320 gtggggggcc accgaggaga aagggaagac ctcagaagtg ctgaacgctg ccaaggtgct 1380 tctcattgag acacagagat gcaacagcag atatgtctat gacaacctga tcacaccagc 1440 catgatctgt gccggcttcc tgcaggggaa cgtcgattct tgccagggtg acagtggagg 1500 gcctctggtc acttcgaaga acaatatctg gtggctgata ggggatacaa gctggggttc 1560 tggctgtgcc aaagcttaca gaccaggagt gtacgggaat gtgatggtat tcacggactg 1620 gatttatcga caaatgaggg cagacggcta atccacatgg tcttcgtcct tgacgtcgtt 1680 ttacaagaaa acaatggggc tggttttgct tccccgtgca tgatttactc ttagagatga 1740 ttcagaggtc acttcatttt tattaaacag tgaacttgtc tggctttggc actctctgcc 1800 attctgtgca ggctgcagtg gctcccctgc ccagcctgct ctccctaacc ccttgtccgc 1860 aaggggtgat ggccggctgg ttgtgggcac tggcggtcaa gtgtggagga gaggggtgga 1920 ggctgcccca ttgagatctt cctgctgagt cctttccagg ggccaatttt ggatgagcat 1980 ggagctgtca cctctcagct gctggatgac ttgagatgaa aaaggagaga catggaaagg 2040 gagacagcca ggtggcacct gcagcggctg ccctctgggg ccacttggta gtgtccccag 2100 cctacctctc cacaagggga ttttgctgat gggttcttag agccttagca gccctggatg 2160 gtggccagaa ataaagggac cagcccttca tgggtggtga cgtggtagtc acttgtaagg 2220 ggaacagaaa catttttgtt cttatggggt gagaatatag acagtgccct tggtgcgagg 2280 gaagcaattg aaaaggaact tgccctgagc actcctggtg caggtctcca cctgcacatt 2340 gggtggggct cctgggaggg agactcagcc ttcctcctca tcctccctga ccctgctcct 2400 agcaccctgg agagtgcaca tgccccttgg tcctggcagg gcgccaagtc tggcaccatg 2460 ttggcctctt caggcctgct agtcactgga aattgaggtc catgggggaa atcaaggatg 2520 ctcagtttaa ggtacactgt ttccatgtta tgtttctaca cattgctacc tcagtgctcc 2580 tggaaactta gcttttgatg tctccaagta gtccaccttc atttaactct ttgaaactgt 2640 atcatctttg ccaagtaaga gtggtggcct atttcagctg ctttgacaaa atgactggct 2700 cctgacttaa cgttctataa atgaatgtgc tgaagcaaag tgcccatggt ggcggcgaag 2760 aagagaaaga tgtgttttgt tttggactct ctgtggtccc ttccaatgct gtgggtttcc 2820 aaccagggga agggtccctt ttgcattgcc aagtgccata accatgagca ctactctacc 2880 atggttctgc ctcctggcca agcaggctgg tttgcaagaa tgaaatgaat gattctacag 2940 ctaggactta accttgaaat ggaaagtcat gcaatcccat ttgcaggatc tgtctgtgca 3000 catgcctctg tagagagcag cattcccagg gaccttggaa acagttggca ctgtaaggtg 3060 cttgctcccc aagacacatc ctaaaaggtg ttgtaatggt gaaaacgtct tccttcttta 3120 ttgccccttc ttatttatgt gaacaactgt ttgtcttttt ttgtatcttt tttaaactgt 3180 aaagttcaat tgtgaaaatg aatatcatgc aaataaatta tgcaattttt ttttcaaagt 3240 aaaaaaaaaa 3250 <210>    3 <211> 3175 <212> DNA <213> Mus musculus <400>    3 gcctttcctg gccgttccct ccttctggcc gaggtgcctg cgtttagggg tgtcaccctg 60 gctcccggga cgccgcctcc ggagatttaa gcgagaactg gagtaggtcg tgtacttgga 120 gcggacgagg aagccaagag ctcggacaga ggcggagagg ggcgggaagc gcaacaggtc 180 acctggagga agccccatac tgacctcctc atgctgctga cacaggcagg atggcattga 240 actcagggtc acctccagga atcggacctt gctatgagaa ccacgggtat cagtctgagc 300 acatctgtcc tccgagacca ccagtggctc ccaatggcta caacttgtat ccagcccagt 360 actacccatc tccagtgcct cagtatgctc cgaggattac aacgcaagcc tcaacatctg 420 tcatccacac acatcccaag tcctcaggag cactgtgcac ctcaaagtct aagaaatcgc 480 tgtgtttagc cctcgccctg ggcactgtcc tcacgggagc tgctgtggct gctgtcttgc 540 tttggaggtt ctgggacagc aactgttcta cgtctgagat ggagtgtggg tcttcaggca 600 catgcatcag ctcttctctc tggtgtgacg gggtagcaca ttgtcccaac ggagaagatg 660 agaaccgttg tgttcgtctc tacggacaaa gcttcatcct ccaggtttac tcatctcaga 720 ggaaagcctg gtatcccgtg tgccaggatg attggagtga gagctacggg agagcagcat 780 gtaaagacat gggatacaag aacaattttt attctagcca agggatacca gaccagagcg 840 gggcaacgag ctttatgaag ctgaatgtga gctcaggcaa cgttgacctc tataaaaaac 900 tctaccacag tgactcatgt tcatcccgca tggtggtttc tttgcgctgt atagaatgcg 960 gggttcgctc agtgaaacgc cagagcagga ttgtgggtgg attgaatgcc tcaccaggag 1020 actggccctg gcaggtcagc ctgcacgtcc aaggcgtcca cgtctgcgga ggctccatca 1080 tcacccccga gtggattgtg acggccgccc actgtgtgga agaacccctc agcagcccga 1140 ggtactggac ggcatttgcg ggaattctga gacagtctct catgttctat ggaagtagac 1200 accaggtaga aaaagtaatt tcccatccaa attacgactc taagaccaag aataacgaca 1260 ttgctctcat gaagctgcag acacctttgg cttttaatga tctagtgaag ccagtgtgtc 1320 tgccgaaccc aggcatgatg ctagacctag accaggaatg ctggatttcg gggtgggggg 1380 ccacctatga gaaagggaag acctcggacg tgttgaatgc tgccatggta cccttgatcg 1440 agccctccaa atgtaatagt aaatacatat acaacaacct aatcacacca gccatgatct 1500 gtgccggctt cctccagggg tctgtcgact cttgccaggg agacagtgga gggccgctgg 1560 ttactttgaa gaatgggatc tggtggctga ttggggacac gagctggggc tcgggctgtg 1620 ccaaggcact cagacctgga gtatacggga acgtgacggt atttacagat tggatctacc 1680 agcaaatgag ggcgaacagc taatccacgt ggctttgtcc cagacttcct ttgtcttcaa 1740 caaccttctg caagaaaacc aagggcctga attttaactt cctgtgcaca atgtaccttt 1800 tgagatgatt cgaagggcct ttcactttta ttaaacagtg acttgtttga ctgtgctccc 1860 tggtcctgtg agggcttcag tgccccaccc ctgggccact tctgcagctc ccaccagaat 1920 ggatgaccag attctgttgg gtttgggcac atagggccaa aggcagagga gggtggcact 1980 ctcatgttgg aacttctttt gggctcatgc tcaggccttt tttggatcac taaggactat 2040 gacctctgag taacctgatg acctgagaaa gagtaaggag gccaggcagg gccttgggcc 2100 caggaacagg taccttgaga gtgagagcta cccattgcct gtggcctaaa tctgctgtgc 2160 aggttgggct ggtcatactg tcatgatttc attaacagcc tgggtgaaca tggctgggag 2220 taaagggctt gctctcctgc atgttgacat gacggccctt tccaagggtg atggaggctt 2280 tcccaagcta agggcctagg cagatctctc agagcaagaa gctaatgccg gcatgtccct 2340 tgggtgagct ctacatggtg ttattcagtc tggttcttgg ctccccacta ctgtttctct 2400 cagcctctca gagcctgaaa cttacctctt agctttggct acaggcatgg cctagtacct 2460 gatggagcct gtatagctca gctaatcaaa tggaggctca ggtccatcag aatcagggac 2520 ttgtgatttc agtcaccttg cttctgggtt gtgtttcttc tcttactacc tcactgcacc 2580 tggacactag agtggatgaa tgtctggagt tcacctgcat ttggactgtg tgattgtgcc 2640 tcagacacta gacctcttcc agatggttag gttgttctgt agactggcaa tgagattaga 2700 agttcctagc ttcagataaa gatgaaagag aggagatcat tgtcttctgt cttcttctgg 2760 ccctgggttt ataccaggaa agccatgcca gaattaccaa atatgaagta tgaatgtctt 2820 acccacggtg aggctctgcc tccttctctc tgcctggttc ttcagaaggc agtgaatggg 2880 tcataactgg gactccatct ttgctgggga aagtctccca cctagggaat ggttaccact 2940 ccatgtaaag aaaactccct catgcgtcct ctgggacctt cttagatgct gtaaggtacc 3000 tacatacaga ctaaatgtgc aagcaccttg aagtgtgaga acctgtcccc tccttagctc 3060 tccttgtctt tgctgttggt tggttatttc ctgctttgtg tctgttctga gctgtgagat 3120 tccactgtga aatatatgaa taaagtatat aattctttta aaaaaaaaaa aaaaa 3175 <210>    4 <211> 3135 <212> DNA <213> Rattus norvegicus <400>    4 ctggagtagg tcgtgtactt ggagtggaca aggaaaccga gagccccggc ggaggcggag 60 aggggcgggg agcacagcag gtcacctgga ggaagccaca tactgacctc ctcatgctgc 120 tgacacaggc aggatggcat tgaactcagg gtcacctcca ggaattggac cttactatga 180 gaaccacggg tatcagtctg agcacgtcta ttccccgagg ccacctgtgt ctcccagtgg 240 ctacaacttg tatccagccc agagctgccc atctccagtg ccccagtatg ctccgagagt 300 cacaactcaa gcctcaacac ctgccatcca catacagccc cgatcctcag gaacactgtg 360 cacctcaaaa tctaagaaat ccatgcttgt cgccctggcc ctgggcactg tcctcgccgg 420 tgctgctgtg gctgctggct tgctttggaa gttctgggac agcaagtgct cttcatcaga 480 gatggagtgt gggtcttcag ggacatgtat cagctcctcc ctctggtgtg acggagtgtc 540 acagtgtccc aacggggaag acgagaaccg gtgtgttcgc ctctatggaa caagcttcac 600 cctccaggtt tactcatctc agaggaaagc ctggtatccc gtctgccagg atgattggaa 660 tgagagctac gggagagcag catgtaaaga catgggatac aagaacagct tttattctag 720 ccaagggata ccagaccaga gcggggcaac gagctttatg aagctgaatg tgagcgcagg 780 caacgtcgac ctctataaaa aactctacca cagtgactcg tgctcatccc gcatggtggt 840 ttctttgcgc tgtatagaat gcggggttcg ctcagtaaga cgtcagagca ggattgtggg 900 tgggtcgacc gcctcaccag gagactggcc ctggcaggtc agcctgcacg tccaaggtat 960 ccatgtctgc ggaggctcca tcatcacccc cgagtggatt gtgacagccg cccactgtgt 1020 ggaagaaccc ctcagcagcc ctaggtactg gacggcattt gcgggaattt tgaaacagtc 1080 tctcatgttc tatggaagta gacaccaggt agaaaaagtg atttcccatc caaattacga 1140 ctctaagacc aagaataatg acattgctct catgaagctg cagacaccct tggcttttaa 1200 tgatgtagtg aagccagtgt gtctgccgaa cccaggcatg atgctggacc tagcccagga 1260 gtgctggatt tcagggtggg gggccaccta tgagaaaggg aagacctcag atgtgctgaa 1320 tgctgccatg gtacccttga tcgagccctc caaatgtaat agcaaataca tatacaacaa 1380 cttaatcaca ccagccatga tctgcgccgg gttcctccag ggctctgtcg actcttgtca 1440 gggagacagt ggagggcccc tggttacttt gaagaatgaa atctggtggc tgattgggga 1500 cacgagctgg ggctcgggct gtgccaaggc atacagacct ggagtatacg ggaatgtgac 1560 agtatttaca gattggatct accagcaaat gagggcgaac agctaatcca cgtggctttg 1620 tccccgactt cctttgtctt caacaacctt ctgcaagaaa accaaggggc ggattttcaa 1680 cttcctgtgc acaatgtacc ttttgagatg attcaaaggg cctttcactt ttattaaaca 1740 gtgacttatc tgactgtgct ccctggtcct gtgagggctt cagtgcccca ccctccaggc 1800 tacttcagca gctcctacca gaaaggatga ccagatcctg ttgggtttgg gcacataggg 1860 ccaaatacag aagaaggtgg cactctcatg ttgagacttc tttttggctt atgctcaggc 1920 ctcttttgga tgagtaagga ctatgacctc tgagtagtct gatgacctga gaagagtagg 1980 gaagccaggc agggccttgg gccaaggtac aggtacagag agagaaagag agagagagag 2040 agagagagag agagagagag agagagagag agagagagag agagagaaca ctacccattg 2100 cctgtggcct agctctgctg tgtagttttg ggctggtcat actataatga cttcatgaac 2160 aacagcccag gagtaaagga cttattctcc tgcatgttga catgacagtc atttccaagg 2220 ttgatggaac ctttgccagg ctgagggcct aggcagggcc ttcagagcaa gaaactaatg 2280 tctgcatgtc ccttgggtga gctctatatg gtatcattca gtctggttct tggctcccca 2340 ccgctatttc tgtcagcctt tcacagcctg aaacttacct ctttggtttg gctccaggta 2400 aggcctggta cttgatggag cctgtatagc tcagctagtc atctagaggc tgaggtccat 2460 tagaatcagg gacttgtgaa tccagtcacc ttgcttctgg gttgtgttta ttcccttact 2520 acctcactgc acctgcacac tagtctggat gaatgtctgt agttcacctg catttggact 2580 gtgtgcttgt gcctcagata ttagacctct tccagatggt taggttgttc tgtagactga 2640 cgatgggatt agtagtccca agctttggac aaagatgaaa aagaggagct cgttgtcttc 2700 tatccttttc tggccctggg tttataccag gaaagccatg ccacaatcac caaatatgaa 2760 gtatgaatgt cttacctatg gtgaggctct gcctcctcct ctgtgcctgg ctcttcagaa 2820 ggcagtgaat gggtcacagc tgggactccg tctttgctgg ggagggtctc ccaccaagga 2880 aatagttgcc actccatgta aagagttccc tcatgcttcc tctgggaaca acactctagg 2940 gaccttctta gatgccataa ggtacctact tacaagacta aatgtacaag caccttgaag 3000 tgtgagaaca tgttccctcc ttagctctcc ctgtctttgc tgttggttgg ttatttcctg 3060 ttttgtgtct gttctgagct gtgagattcc actgtgaaat gtatgaataa agtatgtaat 3120 tctgtccatt gttca 3135 <210>    5 <211> 3609 <212> DNA <213> Macaca fascicularis <400>    5 gtgagtcacc acgcccgacc cagctggaga acgttattgg cagcatcact ctggtcttcc 60 atttcaaagt ccatggtagc tgatttgggc acgctgaata tttttgttta tcatgttctg 120 attgctggaa gagataggat tatcctgcca ctgcctctgg gtcccgtggc ttttatttga 180 caattaatgg tggtggctcc cggtgttcct tccttccctg gcttccttgt tagggatcgt 240 gaatgaatag caagtgtggg ttttgagcac tgattcttca ttcctttcag agggcttctg 300 agctgggaga gccgccaatg ggatcgaatt ttcacttcct gaaacttcaa ccaaacctat 360 aactggggag ataggagagc tgagaaacca gaggcgtgag aatgtgtttg gttaggcagg 420 actctttgaa agcaatctta ggacggtact gagttctctc ctgcttatga gacaagaatg 480 cgggatttat tgtgttgatg tcaggcctga ggatcctctc tcttcacaaa gcagacatag 540 gtcatactga acattccaga tacctatcat tactcgatgc tgttgataaa agcaagatgg 600 ctttgaactc agggtcaccg ccaggtgttg gaccttacta cgaaaaccat ggataccaac 660 cggaaaaccc ctatcctgca cagcccaccg tggcccccaa tgtctacgag gtgcatccgg 720 ctcagtacta cccgtccccc gtaccccagt acaccccgag ggtcctgacg catgcttcca 780 accccgccgt ctgcaggcag cccaaatccc cgtcggggac agtgtgcacc tcaaagacta 840 agaaagcact gtgcgtcacc atgaccctgg gggccgtcct cgtgggagct gcgctggccg 900 ctggcctgct ctggaagttc atgggcagca agtgctccga ctctgggata gagtgcgact 960 cctcaggtac ctgcatcagc tcctctaact ggtgcgatgg cgtgtcacac tgcccgaacg 1020 gggaggacga gaaccggtgt gttcgcctct atggaccaaa cttcattctt caggtgtact 1080 catctcagag gaagtcctgg caccctgtat gccgagacga ctggaacgag aactacgcgc 1140 gggcagcctg cagggacatg ggctataaga atagttttta ctctagccaa ggaatagtgg 1200 ataacagtgg agccaccagc tttatgaaac tgaacacaag tgctggcaat gtcgatatct 1260 ataaaaaact gtaccacagt gatgcctgtt cttcaaaagc agtggtttct ttacgctgta 1320 tagcttgtgg ggtccgctca aacttaaacc gccagagcag gatcgtgggc ggccagaacg 1380 cgctcctggg ggcctggccc tggcaggtca gtctgcacgt ccagaacatc catgtgtgcg 1440 gaggctccat catcaccccc gagtggatcg tgacagctgc tcactgcgtg gaaaaacctc 1500 ttaacagtcc gtggcaatgg acggcatttg tggggacttt gagacaatct tccatgttct 1560 atgaaaaagg acaccgagta gaaaaagtga tttctcatcc aaattatgac tccaagacca 1620 agaacaatga cattgcgctg atgaagctgc atacgcctct gactttcaac gaggtggtga 1680 aaccagtatg tctgcccaac ccaggcatga tgctggagcc ggaacagcac tgctggattt 1740 ctgggtgggg ggccacccag gagaaaggga agacctcaga cgtgctgaac gctgccatgg 1800 tgcctctcat tgagccgcgg agatgcaaca acaaatacat ctacgacggc ctgatcacac 1860 cagccatgat ctgtgccgga ttcctgcggg ggaccgttga ttcttgccag ggtgacagtg 1920 gagggcctct ggtcactttg aagaacgatg tctggtggct gatcggggat acaagctggg 1980 gatctggctg tgcccaagct aacagaccag gagtgtacgg gaatgtgacc gtcttcacgg 2040 actggattta tcgacaaatg agggcagatg actaatcctc atggtctttg tccttgacgt 2100 cgttctacga gaaaacaaag cggctggttt tgcttccccg tgcgtgattt actcttacag 2160 atgattcaga ggtcacttca tttttattaa acagtgaact tgcctggctt tggcactctc 2220 tgccattctg tgtagtctgc agtggctcca ctgcccagcc tgttctcctt aaccccttgt 2280 ccacaagggg tgatggccgg ctggttgtgg acactggtgg tcaagtgtga aggagagggg 2340 tggaggctgc cccattgaga tcttcctttt gagtcctttc caggggccac tttcggatga 2400 gcatggagct gctgcctctc cgctgctgga cttgagatgg aaaaggagag acgtggaaag 2460 ggagacagcc aggtggcacc tgcagcggct gccctctggg gccacttggg tagtgtcccc 2520 agcctagttc tccacagggg gttttggatg attggttctt agtgccttag cagccccgga 2580 tggtggctgg aaataaaggg accagccctt catgggtggt gacgtggtag tcacttgtaa 2640 gggcaacaga aacatttttg tttttacagg gtgagaatgt agacagtgcc cttggtgcaa 2700 gggaagcaat tgcacaggca cttgccctga gcactcctgg tgcaggtctc cacctgcaca 2760 ttgggtgggg ctccctggag attgcacatg ccccttggtc ctggcagggg gccaagtctg 2820 gcaccacgtt ggcctcttca ggcctactag tcactggaaa ttgaggtcca tgggggaaat 2880 caaggatgct cagttttagg tacactgttt ccaagttatg tttctacaca ttgctacctc 2940 aatgctcctg gaatcttagc atttgatgtc tccaagtagt ccacctacat ttaactcttt 3000 gaaactatca tctttttgcc aagtttgagt ggtggcctat ttgagatact ttgacaaaat 3060 gattggcttc tgactttatg ttctataaat gaatgtgctg aaggaaagtg cccatggtgg 3120 cggtgaagaa gagaaagatg tgttttgttt tggactctct gtggttcctt ccaatgccgt 3180 gggtttccaa ccaggggaag ggtccctctt gtattaccaa gtgccataac catgagcact 3240 actctagcat ggttctgcct cttggccaag caggctggtt tgcaagaatg aaatgaatga 3300 ttctactgct aggacttaac cttgaaatgg aaggtcatgc aatcccattt gcaggatctg 3360 tctgtgcacg tgcctctgta gagagcagca ttcccaggca ccttggaaac aggtggcact 3420 ataaggtgcc tgctccccaa gacacatcct aaaaggtgtt ataatggtga aaacgtcttc 3480 cttctttatt gccccttttt atttatgtga gcaaccattt gtcttttttg tatctttttt 3540 aaactgtaaa gttcaattgt gaaaatgaat atcatgcaaa taaattatgc aatttttttt 3600 tcaaagtaa 3609 <210>    6 <211> 3450 <212> DNA <213> Homo sapiens <400>    6 taaattgcag tcttgggtat gtctttatta ggagcatgga aacagactaa tagaataata 60 agaaggagtc atttgaggga cctctccaac aggacaggct gcaggaactg agggcaccaa 120 ccacaggagc accaagtctg gccttgcctc acctttggtc ctctgaccag cagcctcaac 180 atcaccccct tataaggaaa tggaggctgg tcctactcac caggcagaac ttcaaagatg 240 cagtagttac tttgaaaaaa aaattgcata atttatttgc atgatattca ttttcacaat 300 tgaactttac agtttaaaaa agatacaaaa aaagacaaac agttgttcac ataaataaga 360 aggggcaata aagaaggaag acgttttcac cattacaaca ccttttagga tgtgtcttgg 420 ggagcaagca ccttacagtg ccaactgttt ccaaggtccc tgggaatgct gctctctaca 480 gaggcatgtg cacagacaga tcctgcaaat gggattgcat gactttccat ttcaaggtta 540 agtcctagct gtagaatcat tcatttcatt cttgcaaacc agcctgcttg gccaggaggc 600 agaaccatgg tagagtagtg ctcatggtta tggcacttgg caatgcaaaa gggacccttc 660 ccctggttgg aaacccacag cattggaagg gaccacagag agtccaaaac aaaacacatc 720 tttctcttct tcgccgccac catgggcact ttgcttcagc acattcattt atagaacgtt 780 aagtcaggag ccagtcattt tgtcaaagca gctgaaatag gccaccactc ttacttggca 840 aagatgatac agtttcaaag agttaaatga aggtggacta cttggagaca tcaaaagcta 900 agtttccagg agcactgagg tagcaatgtg tagaaacata acatggaaac agtgtacctt 960 aaactgagca tccttgattt cccccatgga cctcaatttc cagtgactag caggcctgaa 1020 gaggccaaca tggtgccaga cttggcgccc tgccaggacc aaggggcatg tgcactctcc 1080 agggtgctag gagcagggtc agggaggatg aggaggaagg ctgagtctcc ctcccaggag 1140 ccccacccaa tgtgcaggtg gagacctgca ccaggagtgc tcagggcaag ttccttttca 1200 attgcttccc tcgcaccaag ggcactgtct atattctcac cccataagaa caaaaatgtt 1260 tctgttcccc ttacaagtga ctaccacgtc accacccatg aagggctggt ccctttattt 1320 ctggccacca tccagggctg ctaaggctct aagaacccat cagcaaaatc cccttgtgga 1380 gaggtaggct ggggacacta ccaagtggcc ccagagggca gccgctgcag gtgccacctg 1440 gctgtctccc tttccatgtc tctccttttt catctcaagt catccagcag ctgagaggtg 1500 acagctccat gctcatccaa aattggcccc tggaaaggac tcagcaggaa gatctcaatg 1560 gggcagcctc cacccctctc ctccacactt gaccgccagt gcccacaacc agccggccat 1620 caccccttgc ggacaagggg ttagggagag caggctgggc aggggagcca ctgcagcctg 1680 cacagaatgg cagagagtgc caaagccaga caagttcact gtttaataaa aatgaagtga 1740 cctctgaatc atctctaaga gtaaatcatg cacggggaag caaaaccagc cccattgttt 1800 tcttgtaaaa cgacgtcaag gacgaagacc atgtggatta gccgtctgcc ctcatttgtc 1860 gataaatcca gtccgtgaat accatcacat tcccgtacac tcctggtctg taagctttgg 1920 cacagccaga accccagctt gtatccccta tcagccacca gatattgttc ttcgaagtga 1980 ccagaggccc tccactgtca ccctggcaag aatcgacgtt cccctgcagg aagccggcac 2040 agatcatggc tggtgtgatc aggttgtcat agacatatct gctgttgcat ctctgtgtct 2100 caatgagaag caccttggca gcgttcagca cttctgaggt cttccctttc tcctcggtgg 2160 ccccccaccc ggaaatccag cagagctgtt ctggctgcag catcatgcct gggttgggca 2220 gacacactgg tttcactagg tcgttgaaag tcagaggctt ctgcagcttc atcagcgcaa 2280 tgtcattgtt cttggtcttg gagtcataat ttggatgaga aatcactttt tctacttggt 2340 atccggctcc atagaacatg aaagattgtc tcaaaatccc cgcaaatgcc gtccaatgcc 2400 atggattgtt aagaggtttt tccacgcagt gggcggctgt cacgatccac tcgggggtga 2460 tgatggagcc tccgcacacg tggacgttct ggacgtgcag gctgacctgc cagggccagg 2520 cccccgggag cgcgctctcg ccgcccacaa tcctgctctg gcggcttgag ttcaagttga 2580 ccccgcaggc tatacagcgt aaagaaacca ctgcttttga agaacaggca tcactgtggt 2640 acagtttttt atagatatcg acattgccgg cacttgtgtt cagtttcata aagctggtgg 2700 atccgctgtc atccactatt ccttggctag agtaaaaatt attcttatag cccatgtccc 2760 tgcaggccgc ccgcccgtag ttctcgttcc agtcgtcttg gcacacaggg tgccaggact 2820 tcctctgaga tgagtacacc tgaaggatga agtttggtcc gtagaggcga acacaccgat 2880 tctcgtcctc cccgccgggg cagtgtgaca cgccatcaca ccagttagag gggttgatgc 2940 aggtacctga ggagtcgcac tctatcccag agttggagca cttgctgccc atgaacttcc 3000 agagtaggcc agcggccagc gcagctccca cgaggaaggt ccccagggtc aaggtgatgc 3060 acagtgcttt cttagtcttt gaggtgcaca ctgtcccgga tggggatttg ggctgcgtgc 3120 agacgacggg gttggaagcc tgcgtcagga ccctcggggc gtactggggc acgggggacg 3180 ggtagtactg agccggatgc acctcgtaga cagtggggac cacagtgggc tgtgcgggat 3240 aggggttttc cggttggtat ccatggtttt catagtaagg tccaatagct ggtggtgacc 3300 ctgagttcaa agccatcttg ctgttatcaa cagcatcgag taatgatagg tatctggaat 3360 gttcaatatg acctgccgcg ctccaggcgg cgctccccgc ccctcgccct ccgcctccgc 3420 ctccgcctcc tgcttagctc gcgcctactc 3450 <210>    7 <211> 3250 <212> DNA <213> Homo sapiens <400>    7 tttttttttt actttgaaaa aaaaattgca taatttattt gcatgatatt cattttcaca 60 attgaacttt acagtttaaa aaagatacaa aaaaagacaa acagttgttc acataaataa 120 gaaggggcaa taaagaagga agacgttttc accattacaa caccttttag gatgtgtctt 180 ggggagcaag caccttacag tgccaactgt ttccaaggtc cctgggaatg ctgctctcta 240 cagaggcatg tgcacagaca gatcctgcaa atgggattgc atgactttcc atttcaaggt 300 taagtcctag ctgtagaatc attcatttca ttcttgcaaa ccagcctgct tggccaggag 360 gcagaaccat ggtagagtag tgctcatggt tatggcactt ggcaatgcaa aagggaccct 420 tcccctggtt ggaaacccac agcattggaa gggaccacag agagtccaaa acaaaacaca 480 tctttctctt cttcgccgcc accatgggca ctttgcttca gcacattcat ttatagaacg 540 ttaagtcagg agccagtcat tttgtcaaag cagctgaaat aggccaccac tcttacttgg 600 caaagatgat acagtttcaa agagttaaat gaaggtggac tacttggaga catcaaaagc 660 taagtttcca ggagcactga ggtagcaatg tgtagaaaca taacatggaa acagtgtacc 720 ttaaactgag catccttgat ttcccccatg gacctcaatt tccagtgact agcaggcctg 780 aagaggccaa catggtgcca gacttggcgc cctgccagga ccaaggggca tgtgcactct 840 ccagggtgct aggagcaggg tcagggagga tgaggaggaa ggctgagtct ccctcccagg 900 agccccaccc aatgtgcagg tggagacctg caccaggagt gctcagggca agttcctttt 960 caattgcttc cctcgcacca agggcactgt ctatattctc accccataag aacaaaaatg 1020 tttctgttcc ccttacaagt gactaccacg tcaccaccca tgaagggctg gtccctttat 1080 ttctggccac catccagggc tgctaaggct ctaagaaccc atcagcaaaa tccccttgtg 1140 gagaggtagg ctggggacac taccaagtgg ccccagaggg cagccgctgc aggtgccacc 1200 tggctgtctc cctttccatg tctctccttt ttcatctcaa gtcatccagc agctgagagg 1260 tgacagctcc atgctcatcc aaaattggcc cctggaaagg actcagcagg aagatctcaa 1320 tggggcagcc tccacccctc tcctccacac ttgaccgcca gtgcccacaa ccagccggcc 1380 atcacccctt gcggacaagg ggttagggag agcaggctgg gcaggggagc cactgcagcc 1440 tgcacagaat ggcagagagt gccaaagcca gacaagttca ctgtttaata aaaatgaagt 1500 gacctctgaa tcatctctaa gagtaaatca tgcacgggga agcaaaacca gccccattgt 1560 tttcttgtaa aacgacgtca aggacgaaga ccatgtggat tagccgtctg ccctcatttg 1620 tcgataaatc cagtccgtga ataccatcac attcccgtac actcctggtc tgtaagcttt 1680 ggcacagcca gaaccccagc ttgtatcccc tatcagccac cagatattgt tcttcgaagt 1740 gaccagaggc cctccactgt caccctggca agaatcgacg ttcccctgca ggaagccggc 1800 acagatcatg gctggtgtga tcaggttgtc atagacatat ctgctgttgc atctctgtgt 1860 ctcaatgaga agcaccttgg cagcgttcag cacttctgag gtcttccctt tctcctcggt 1920 ggccccccac ccggaaatcc agcagagctg ttctggctgc agcatcatgc ctgggttggg 1980 cagacacact ggtttcacta ggtcgttgaa agtcagaggc ttctgcagct tcatcagcgc 2040 aatgtcattg ttcttggtct tggagtcata atttggatga gaaatcactt tttctacttg 2100 gtatccggct ccatagaaca tgaaagattg tctcaaaatc cccgcaaatg ccgtccaatg 2160 ccatggattg ttaagaggtt tttccacgca gtgggcggct gtcacgatcc actcgggggt 2220 gatgatggag cctccgcaca cgtggacgtt ctggacgtgc aggctgacct gccagggcca 2280 ggcccccggg agcgcgctct cgccgcccac aatcctgctc tggcggcttg agttcaagtt 2340 gaccccgcag gctatacagc gtaaagaaac cactgctttt gaagaacagg catcactgtg 2400 gtacagtttt ttatagatat cgacattgcc ggcacttgtg ttcagtttca taaagctggt 2460 ggatccgctg tcatccacta ttccttggct agagtaaaaa ttattcttat agcccatgtc 2520 cctgcaggcc gcccgcccgt agttctcgtt ccagtcgtct tggcacacag ggtgccagga 2580 cttcctctga gatgagtaca cctgaaggat gaagtttggt ccgtagaggc gaacacaccg 2640 attctcgtcc tccccgccgg ggcagtgtga cacgccatca caccagttag aggggttgat 2700 gcaggtacct gaggagtcgc actctatccc agagttggag cacttgctgc ccatgaactt 2760 ccagagtagg ccagcggcca gcgcagctcc cacgaggaag gtccccaggg tcaaggtgat 2820 gcacagtgct ttcttagtct ttgaggtgca cactgtcccg gatggggatt tgggctgcgt 2880 gcagacgacg gggttggaag cctgcgtcag gaccctcggg gcgtactggg gcacggggga 2940 cgggtagtac tgagccggat gcacctcgta gacagtgggg accacagtgg gctgtgcggg 3000 ataggggttt tccggttggt atccatggtt ttcatagtaa ggtccaatag ctggtggtga 3060 ccctgagttc aaagccatct tgctgttatc aacagcatcg agtaatgata ggtatctgga 3120 atgttcaata tgacctgccg cgccgcgctc ctcacacccg ctttcacctc cgggcggggc 3180 agggggcatc ggcgggtccc aggcgcccag gttcccctcc ccagcccgga ccccgagccg 3240 ggaccctggt 3250 <210>    8 <211> 3175 <212> DNA <213> Mus musculus <400>    8 tttttttttt ttttttaaaa gaattatata ctttattcat atatttcaca gtggaatctc 60 acagctcaga acagacacaa agcaggaaat aaccaaccaa cagcaaagac aaggagagct 120 aaggagggga caggttctca cacttcaagg tgcttgcaca tttagtctgt atgtaggtac 180 cttacagcat ctaagaaggt cccagaggac gcatgaggga gttttcttta catggagtgg 240 taaccattcc ctaggtggga gactttcccc agcaaagatg gagtcccagt tatgacccat 300 tcactgcctt ctgaagaacc aggcagagag aaggaggcag agcctcaccg tgggtaagac 360 attcatactt catatttggt aattctggca tggctttcct ggtataaacc cagggccaga 420 agaagacaga agacaatgat ctcctctctt tcatctttat ctgaagctag gaacttctaa 480 tctcattgcc agtctacaga acaacctaac catctggaag aggtctagtg tctgaggcac 540 aatcacacag tccaaatgca ggtgaactcc agacattcat ccactctagt gtccaggtgc 600 agtgaggtag taagagaaga aacacaaccc agaagcaagg tgactgaaat cacaagtccc 660 tgattctgat ggacctgagc ctccatttga ttagctgagc tatacaggct ccatcaggta 720 ctaggccatg cctgtagcca aagctaagag gtaagtttca ggctctgaga ggctgagaga 780 aacagtagtg gggagccaag aaccagactg aataacacca tgtagagctc acccaaggga 840 catgccggca ttagcttctt gctctgagag atctgcctag gcccttagct tgggaaagcc 900 tccatcaccc ttggaaaggg ccgtcatgtc aacatgcagg agagcaagcc ctttactccc 960 agccatgttc acccaggctg ttaatgaaat catgacagta tgaccagccc aacctgcaca 1020 gcagatttag gccacaggca atgggtagct ctcactctca aggtacctgt tcctgggccc 1080 aaggccctgc ctggcctcct tactctttct caggtcatca ggttactcag aggtcatagt 1140 ccttagtgat ccaaaaaagg cctgagcatg agcccaaaag aagttccaac atgagagtgc 1200 caccctcctc tgcctttggc cctatgtgcc caaacccaac agaatctggt catccattct 1260 ggtgggagct gcagaagtgg cccaggggtg gggcactgaa gccctcacag gaccagggag 1320 cacagtcaaa caagtcactg tttaataaaa gtgaaaggcc cttcgaatca tctcaaaagg 1380 tacattgtgc acaggaagtt aaaattcagg cccttggttt tcttgcagaa ggttgttgaa 1440 gacaaaggaa gtctgggaca aagccacgtg gattagctgt tcgccctcat ttgctggtag 1500 atccaatctg taaataccgt cacgttcccg tatactccag gtctgagtgc cttggcacag 1560 cccgagcccc agctcgtgtc cccaatcagc caccagatcc cattcttcaa agtaaccagc 1620 ggccctccac tgtctccctg gcaagagtcg acagacccct ggaggaagcc ggcacagatc 1680 atggctggtg tgattaggtt gttgtatatg tatttactat tacatttgga gggctcgatc 1740 aagggtacca tggcagcatt caacacgtcc gaggtcttcc ctttctcata ggtggccccc 1800 caccccgaaa tccagcattc ctggtctagg tctagcatca tgcctgggtt cggcagacac 1860 actggcttca ctagatcatt aaaagccaaa ggtgtctgca gcttcatgag agcaatgtcg 1920 ttattcttgg tcttagagtc gtaatttgga tgggaaatta ctttttctac ctggtgtcta 1980 cttccataga acatgagaga ctgtctcaga attcccgcaa atgccgtcca gtacctcggg 2040 ctgctgaggg gttcttccac acagtgggcg gccgtcacaa tccactcggg ggtgatgatg 2100 gagcctccgc agacgtggac gccttggacg tgcaggctga cctgccaggg ccagtctcct 2160 ggtgaggcat tcaatccacc cacaatcctg ctctggcgtt tcactgagcg aaccccgcat 2220 tctatacagc gcaaagaaac caccatgcgg gatgaacatg agtcactgtg gtagagtttt 2280 ttatagaggt caacgttgcc tgagctcaca ttcagcttca taaagctcgt tgccccgctc 2340 tggtctggta tcccttggct agaataaaaa ttgttcttgt atcccatgtc tttacatgct 2400 gctctcccgt agctctcact ccaatcatcc tggcacacgg gataccaggc tttcctctga 2460 gatgagtaaa cctggaggat gaagctttgt ccgtagagac gaacacaacg gttctcatct 2520 tctccgttgg gacaatgtgc taccccgtca caccagagag aagagctgat gcatgtgcct 2580 gaagacccac actccatctc agacgtagaa cagttgctgt cccagaacct ccaaagcaag 2640 acagcagcca cagcagctcc cgtgaggaca gtgcccaggg cgagggctaa acacagcgat 2700 ttcttagact ttgaggtgca cagtgctcct gaggacttgg gatgtgtgtg gatgacagat 2760 gttgaggctt gcgttgtaat cctcggagca tactgaggca ctggagatgg gtagtactgg 2820 gctggataca agttgtagcc attgggagcc actggtggtc tcggaggaca gatgtgctca 2880 gactgatacc cgtggttctc atagcaaggt ccgattcctg gaggtgaccc tgagttcaat 2940 gccatcctgc ctgtgtcagc agcatgagga ggtcagtatg gggcttcctc caggtgacct 3000 gttgcgcttc ccgcccctct ccgcctctgt ccgagctctt ggcttcctcg tccgctccaa 3060 gtacacgacc tactccagtt ctcgcttaaa tctccggagg cggcgtcccg ggagccaggg 3120 tgacacccct aaacgcaggc acctcggcca gaaggaggga acggccagga aaggc 3175 <210>    9 <211> 3135 <212> DNA <213> Rattus norvegicus <400>    9 tgaacaatgg acagaattac atactttatt catacatttc acagtggaat ctcacagctc 60 agaacagaca caaaacagga aataaccaac caacagcaaa gacagggaga gctaaggagg 120 gaacatgttc tcacacttca aggtgcttgt acatttagtc ttgtaagtag gtaccttatg 180 gcatctaaga aggtccctag agtgttgttc ccagaggaag catgagggaa ctctttacat 240 ggagtggcaa ctatttcctt ggtgggagac cctccccagc aaagacggag tcccagctgt 300 gacccattca ctgccttctg aagagccagg cacagaggag gaggcagagc ctcaccatag 360 gtaagacatt catacttcat atttggtgat tgtggcatgg ctttcctggt ataaacccag 420 ggccagaaaa ggatagaaga caacgagctc ctctttttca tctttgtcca aagcttggga 480 ctactaatcc catcgtcagt ctacagaaca acctaaccat ctggaagagg tctaatatct 540 gaggcacaag cacacagtcc aaatgcaggt gaactacaga cattcatcca gactagtgtg 600 caggtgcagt gaggtagtaa gggaataaac acaacccaga agcaaggtga ctggattcac 660 aagtccctga ttctaatgga cctcagcctc tagatgacta gctgagctat acaggctcca 720 tcaagtacca ggccttacct ggagccaaac caaagaggta agtttcaggc tgtgaaaggc 780 tgacagaaat agcggtgggg agccaagaac cagactgaat gataccatat agagctcacc 840 caagggacat gcagacatta gtttcttgct ctgaaggccc tgcctaggcc ctcagcctgg 900 caaaggttcc atcaaccttg gaaatgactg tcatgtcaac atgcaggaga ataagtcctt 960 tactcctggg ctgttgttca tgaagtcatt atagtatgac cagcccaaaa ctacacagca 1020 gagctaggcc acaggcaatg ggtagtgttc tctctctctc tctctctctc tctctctctc 1080 tctctctctc tctctctctc tctctctctt tctctctctg tacctgtacc ttggcccaag 1140 gccctgcctg gcttccctac tcttctcagg tcatcagact actcagaggt catagtcctt 1200 actcatccaa aagaggcctg agcataagcc aaaaagaagt ctcaacatga gagtgccacc 1260 ttcttctgta tttggcccta tgtgcccaaa cccaacagga tctggtcatc ctttctggta 1320 ggagctgctg aagtagcctg gagggtgggg cactgaagcc ctcacaggac cagggagcac 1380 agtcagataa gtcactgttt aataaaagtg aaaggccctt tgaatcatct caaaaggtac 1440 attgtgcaca ggaagttgaa aatccgcccc ttggttttct tgcagaaggt tgttgaagac 1500 aaaggaagtc ggggacaaag ccacgtggat tagctgttcg ccctcatttg ctggtagatc 1560 caatctgtaa atactgtcac attcccgtat actccaggtc tgtatgcctt ggcacagccc 1620 gagccccagc tcgtgtcccc aatcagccac cagatttcat tcttcaaagt aaccaggggc 1680 cctccactgt ctccctgaca agagtcgaca gagccctgga ggaacccggc gcagatcatg 1740 gctggtgtga ttaagttgtt gtatatgtat ttgctattac atttggaggg ctcgatcaag 1800 ggtaccatgg cagcattcag cacatctgag gtcttccctt tctcataggt ggccccccac 1860 cctgaaatcc agcactcctg ggctaggtcc agcatcatgc ctgggttcgg cagacacact 1920 ggcttcacta catcattaaa agccaagggt gtctgcagct tcatgagagc aatgtcatta 1980 ttcttggtct tagagtcgta atttggatgg gaaatcactt tttctacctg gtgtctactt 2040 ccatagaaca tgagagactg tttcaaaatt cccgcaaatg ccgtccagta cctagggctg 2100 ctgaggggtt cttccacaca gtgggcggct gtcacaatcc actcgggggt gatgatggag 2160 cctccgcaga catggatacc ttggacgtgc aggctgacct gccagggcca gtctcctggt 2220 gaggcggtcg acccacccac aatcctgctc tgacgtctta ctgagcgaac cccgcattct 2280 atacagcgca aagaaaccac catgcgggat gagcacgagt cactgtggta gagtttttta 2340 tagaggtcga cgttgcctgc gctcacattc agcttcataa agctcgttgc cccgctctgg 2400 tctggtatcc cttggctaga ataaaagctg ttcttgtatc ccatgtcttt acatgctgct 2460 ctcccgtagc tctcattcca atcatcctgg cagacgggat accaggcttt cctctgagat 2520 gagtaaacct ggagggtgaa gcttgttcca tagaggcgaa cacaccggtt ctcgtcttcc 2580 ccgttgggac actgtgacac tccgtcacac cagagggagg agctgataca tgtccctgaa 2640 gacccacact ccatctctga tgaagagcac ttgctgtccc agaacttcca aagcaagcca 2700 gcagccacag cagcaccggc gaggacagtg cccagggcca gggcgacaag catggatttc 2760 ttagattttg aggtgcacag tgttcctgag gatcggggct gtatgtggat ggcaggtgtt 2820 gaggcttgag ttgtgactct cggagcatac tggggcactg gagatgggca gctctgggct 2880 ggatacaagt tgtagccact gggagacaca ggtggcctcg gggaatagac gtgctcagac 2940 tgatacccgt ggttctcata gtaaggtcca attcctggag gtgaccctga gttcaatgcc 3000 atcctgcctg tgtcagcagc atgaggaggt cagtatgtgg cttcctccag gtgacctgct 3060 gtgctccccg cccctctccg cctccgccgg ggctctcggt ttccttgtcc actccaagta 3120 cacgacctac tccag 3135 <210>   10 <211> 3609 <212> DNA <213> Macaca fascicularis <400>   10 ttactttgaa aaaaaaattg cataatttat ttgcatgata ttcattttca caattgaact 60 ttacagttta aaaaagatac aaaaaagaca aatggttgct cacataaata aaaaggggca 120 ataaagaagg aagacgtttt caccattata acacctttta ggatgtgtct tggggagcag 180 gcaccttata gtgccacctg tttccaaggt gcctgggaat gctgctctct acagaggcac 240 gtgcacagac agatcctgca aatgggattg catgaccttc catttcaagg ttaagtccta 300 gcagtagaat cattcatttc attcttgcaa accagcctgc ttggccaaga ggcagaacca 360 tgctagagta gtgctcatgg ttatggcact tggtaataca agagggaccc ttcccctggt 420 tggaaaccca cggcattgga aggaaccaca gagagtccaa aacaaaacac atctttctct 480 tcttcaccgc caccatgggc actttccttc agcacattca tttatagaac ataaagtcag 540 aagccaatca ttttgtcaaa gtatctcaaa taggccacca ctcaaacttg gcaaaaagat 600 gatagtttca aagagttaaa tgtaggtgga ctacttggag acatcaaatg ctaagattcc 660 aggagcattg aggtagcaat gtgtagaaac ataacttgga aacagtgtac ctaaaactga 720 gcatccttga tttcccccat ggacctcaat ttccagtgac tagtaggcct gaagaggcca 780 acgtggtgcc agacttggcc ccctgccagg accaaggggc atgtgcaatc tccagggagc 840 cccacccaat gtgcaggtgg agacctgcac caggagtgct cagggcaagt gcctgtgcaa 900 ttgcttccct tgcaccaagg gcactgtcta cattctcacc ctgtaaaaac aaaaatgttt 960 ctgttgccct tacaagtgac taccacgtca ccacccatga agggctggtc cctttatttc 1020 cagccaccat ccggggctgc taaggcacta agaaccaatc atccaaaacc ccctgtggag 1080 aactaggctg gggacactac ccaagtggcc ccagagggca gccgctgcag gtgccacctg 1140 gctgtctccc tttccacgtc tctccttttc catctcaagt ccagcagcgg agaggcagca 1200 gctccatgct catccgaaag tggcccctgg aaaggactca aaaggaagat ctcaatgggg 1260 cagcctccac ccctctcctt cacacttgac caccagtgtc cacaaccagc cggccatcac 1320 cccttgtgga caaggggtta aggagaacag gctgggcagt ggagccactg cagactacac 1380 agaatggcag agagtgccaa agccaggcaa gttcactgtt taataaaaat gaagtgacct 1440 ctgaatcatc tgtaagagta aatcacgcac ggggaagcaa aaccagccgc tttgttttct 1500 cgtagaacga cgtcaaggac aaagaccatg aggattagtc atctgccctc atttgtcgat 1560 aaatccagtc cgtgaagacg gtcacattcc cgtacactcc tggtctgtta gcttgggcac 1620 agccagatcc ccagcttgta tccccgatca gccaccagac atcgttcttc aaagtgacca 1680 gaggccctcc actgtcaccc tggcaagaat caacggtccc ccgcaggaat ccggcacaga 1740 tcatggctgg tgtgatcagg ccgtcgtaga tgtatttgtt gttgcatctc cgcggctcaa 1800 tgagaggcac catggcagcg ttcagcacgt ctgaggtctt ccctttctcc tgggtggccc 1860 cccacccaga aatccagcag tgctgttccg gctccagcat catgcctggg ttgggcagac 1920 atactggttt caccacctcg ttgaaagtca gaggcgtatg cagcttcatc agcgcaatgt 1980 cattgttctt ggtcttggag tcataatttg gatgagaaat cactttttct actcggtgtc 2040 ctttttcata gaacatggaa gattgtctca aagtccccac aaatgccgtc cattgccacg 2100 gactgttaag aggtttttcc acgcagtgag cagctgtcac gatccactcg ggggtgatga 2160 tggagcctcc gcacacatgg atgttctgga cgtgcagact gacctgccag ggccaggccc 2220 ccaggagcgc gttctggccg cccacgatcc tgctctggcg gtttaagttt gagcggaccc 2280 cacaagctat acagcgtaaa gaaaccactg cttttgaaga acaggcatca ctgtggtaca 2340 gttttttata gatatcgaca ttgccagcac ttgtgttcag tttcataaag ctggtggctc 2400 cactgttatc cactattcct tggctagagt aaaaactatt cttatagccc atgtccctgc 2460 aggctgcccg cgcgtagttc tcgttccagt cgtctcggca tacagggtgc caggacttcc 2520 tctgagatga gtacacctga agaatgaagt ttggtccata gaggcgaaca caccggttct 2580 cgtcctcccc gttcgggcag tgtgacacgc catcgcacca gttagaggag ctgatgcagg 2640 tacctgagga gtcgcactct atcccagagt cggagcactt gctgcccatg aacttccaga 2700 gcaggccagc ggccagcgca gctcccacga ggacggcccc cagggtcatg gtgacgcaca 2760 gtgctttctt agtctttgag gtgcacactg tccccgacgg ggatttgggc tgcctgcaga 2820 cggcggggtt ggaagcatgc gtcaggaccc tcggggtgta ctggggtacg ggggacgggt 2880 agtactgagc cggatgcacc tcgtagacat tgggggccac ggtgggctgt gcaggatagg 2940 ggttttccgg ttggtatcca tggttttcgt agtaaggtcc aacacctggc ggtgaccctg 3000 agttcaaagc catcttgctt ttatcaacag catcgagtaa tgataggtat ctggaatgtt 3060 cagtatgacc tatgtctgct ttgtgaagag agaggatcct caggcctgac atcaacacaa 3120 taaatcccgc attcttgtct cataagcagg agagaactca gtaccgtcct aagattgctt 3180 tcaaagagtc ctgcctaacc aaacacattc tcacgcctct ggtttctcag ctctcctatc 3240 tccccagtta taggtttggt tgaagtttca ggaagtgaaa attcgatccc attggcggct 3300 ctcccagctc agaagccctc tgaaaggaat gaagaatcag tgctcaaaac ccacacttgc 3360 tattcattca cgatccctaa caaggaagcc agggaaggaa ggaacaccgg gagccaccac 3420 cattaattgt caaataaaag ccacgggacc cagaggcagt ggcaggataa tcctatctct 3480 tccagcaatc agaacatgat aaacaaaaat attcagcgtg cccaaatcag ctaccatgga 3540 ctttgaaatg gaagaccaga gtgatgctgc caataacgtt ctccagctgg gtcgggcgtg 3600 gtgactcac 3609 

1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of transmembrane serine protease 2 (TMPRSS2) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:6; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.
 2. (canceled)
 3. (canceled)
 4. The dsRNA agent of claim 1, wherein the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-3. 5.-10. (canceled)
 11. The dsRNA agent of claim 1, wherein at least one nucleotide comprises a nucleotide modification.
 12. (canceled)
 13. The dsRNA agent of claim 11, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
 14. The dsRNA agent of any claim 11, wherein at least one of the nucleotide modifications is selected from the group a deoxy-nucleotide modification, a 3′-terminal deoxy-thymine (dT) nucleotide modification, a 2′-O-methyl nucleotide modification, a 2′-fluoro nucleotide modification, a 2′-deoxy nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2′-amino nucleotide modification, a 2′-O-allyl nucleotide modification, 2′-C-alkyl-modified nucleotide modification, a 2′-methoxyethyl nucleotide modification, a 2′-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramidate modification, a non-natural base comprising nucleotide modification, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide comprising a 5′-phosphorothioate group modification, a nucleotide comprising a 5′-methylphosphonate group modification, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic modification, a nucleotide comprising vinyl phosphonate modification, a nucleotide comprising adenosine-glycol nucleic acid (GNA) modification, a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer modification, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate modification, a nucleotide comprising 2′-deoxythymidine-3′phosphate modification, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate modification, a 2′-0 hexadecyl nucleotide modification, a nucleotide comprising a 2′-phosphate modification, a cytidine-2′-phosphate nucleotide modification, a guanosine-2′-phosphate nucleotide modification, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide modification, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide modification, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide modification, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide modification, a 5′-vinyl phosphonate (VP) modification, a 2′-deoxyadenosine-3′-phosphate nucleotide modification, a 2′-deoxycytidine-3′-phosphate nucleotide modification, a 2′-deoxyguanosine-3′-phosphate nucleotide modification, a 2′-deoxythymidine-3′-phosphate nucleotide modification, a 2′-deoxyuridine nucleotide modification, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group modification; and combinations thereof. 15.-17. (canceled)
 18. The dsRNA agent of claim 14, further comprising at least one phosphorothioate internucleotide linkage.
 19. (canceled)
 20. (canceled)
 21. The dsRNA agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide. 22.-28. (Canceled)
 29. The dsRNA agent of claim 1, wherein each strand is 19-30 nucleotides in length.
 30. (canceled)
 31. (canceled)
 32. The dsRNA agent of claim 1, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand. 33.-41. (canceled)
 42. The dsRNA agent of claim 1, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′ end of each strand. 43.-49. (canceled)
 50. The dsRNA agent of claim 1, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.
 51. The dsRNA agent of claim 50, wherein the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
 52. The dsRNA agent of claim 51, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
 53. The dsRNA agent of claim 52, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. 54.-68. (canceled)
 69. The dsRNA agent of claim 1, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.
 70. (canceled)
 71. The dsRNA agent of claim 1, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.
 72. (canceled)
 73. An isolated cell containing the dsRNA agent of claim
 1. 74. A pharmaceutical composition for inhibiting expression of a TMPRSS2 gene, comprising the dsRNA agent of any one of claim
 1. 75. (canceled)
 76. A device for oral inhalative administration comprising the dsRNA agent of claim
 1. 77. (canceled)
 78. A method of inhibiting expression of a TMPRSS2 gene in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the TMPRSS2 gene, thereby inhibiting expression of the TMPRSS2 gene in the cell. 79.-5. (canceled)
 86. A method of treating a subject having a coronavirus infection, a subject at risk of having a coronavirus infection, or a subject at risk of developing a coronavirus infection, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating said subject. 87.-90. (canceled)
 91. The method of claim 86, wherein the administration of the dsRNA is pulmonary system administration.
 92. (canceled)
 93. (canceled)
 94. The method of claim 86, further comprising administering to the subject an additional agent or a therapy suitable for treatment or prevention of a coronavirus-associated disorder.
 95. (canceled) 